Methods of Detecting Signatures of Disease or Conditions in Bodily Fluids

ABSTRACT

Methods and compositions for diagnosing the presence of a cancer cell in an individual are provided. Methods and compositions for identifying a tumor-specific signature in an individual having cancer are also provided. Methods and compositions for diagnosing the presence of an infectious agent in an individual and/or for identifying an infectious agent-specific signature in an infected individual are provided. Methods and compositions for diagnosing the presence of a disease in an individual are also provided. Methods and compositions for identifying a disease-specific signature in an individual having the disease are also provided.

RELATED U.S. APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/349,670, filed on Jan. 13, 2012 which is a divisional of U.S. patentapplication Ser. No. 12/836,191, filed on Jul. 14, 2010 which is acontinuation of PCT Application number PCT/2009/031395 designating theUnited States and filed Jan. 19, 2009 which claims the benefit of thefiling date of U.S. Provisional patent application Ser. Nos. 61/073,434,filed on Jun. 18, 2008 and 61/022,033, filed on Jan. 18, 2008, each ofwhich is hereby incorporated herein by reference in its entirety for allpurposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under R01 CA143101awarded by the National Institutes of Health. The government has certainrights in this invention.

FIELD

The present invention relates to methods of identifying markers ofconditions such as gender of a fetus or disease such as tumor genomic,proteomic, metabolomic, glycomic, glycoproteomic, lipidomic and/orlipoproteomic signatures in cells obtained from bodily fluids of apatient.

BACKGROUND

Tumors originate from normal cells upon the accumulation of genetic andepigenetic alterations. This multi-step process involves multiplegenetic alterations that lead to the progressive transformation ofnormal cells to a malignant phenotype. These alterations are comprisedof irreversible changes in DNA sequence (e.g., mutations, deletions,translocations) and lead to the activation of oncogenes, inactivation oftumor suppressor genes, and fusion of genes. The stochastic nature ofthese events confers genetic heterogeneity that gives the transformedcells molecular fingerprints (e.g., one or more cellular components suchas DNA, RNA, protein, lipid, carbohydrate, and the like) indicative ofcancer that give them unique phenotypes. Consequently, unique gene sethallmarks/signatures are known to be expressed by various tumors (Perouet al. (2000) Nature 406:747; Lobenhofer et al. (2001) Health Perspect.109:881; van't Veer et al. (2002) Nature 415:530 (2002); Liotta and Kohn(2003) Nat. Genet. 33:10; Ginos et al. (2004) Cancer Res. 64:55; Liu(2005) Proc. Natl. Acad. Sci. USA 102:3531; Grigoriadis et al. (2006)Breast Cancer Research 8:R56).

Both primary and metastatic tumors can lie silent and undetected foryears. However, these dormant and occult tumors, as well as previouslydiagnosed primary and metastatic solid tumors, shed daily into thecirculation approximately one-to-six million cells per gram of tumor. Alarge proportion of these circulating tumor cells, known as CTCs,undergo apoptosis and die, whereas distinct cell populations may developinto metastatic disease. Tumor cell apoptotic bodies, DNA, nucleosomes,RNA, and proteins are also found in the blood of cancer patients.Holmgren et al., Blood 93, 3956 (1999). Efforts have been made toinvestigate whether signatures of tumors can be identified and whetherthey can be used to detect or monitor cancer. See, Ransohoff, NatureReviews Cancer 5, 142 (2005) and McLerran et al., Clin. Chem. 54, 44(2008).

DNA can be easily transfected into various eukaryotic cells, i.e., onceit is internalized into the cytoplasm of cells, it is able to integrateits genes into the genome of the host cell. For example, neutrophils andmacrophages can be rapidly and very efficiently (50%-90%) transfected.Passage of DNA from prokaryotic to eukaryotic cells has also beendemonstrated and is believed to occur from eukaryotic to eukaryoticcells. DNA released from tumor cells has a high transforming activity.Adding supernatant medium from cultured tumor cells to normal cellsresults in the appearance of as many transformed foci as those occurringafter a transfection with a cloned ras gene administered as a calciumprecipitate. Furthermore, when healthy rats were injected with plasmafrom tumor-bearing rats (therefore containing tumor DNA) the tumormarker gene was found in the DNA of their lung cells, i.e., tumor geneshave been transcribed in lung cells.

Leukocytes begin as pluripotent hematopoietic stem cells in the bonemarrow and develop along either the myeloid lineage (monocytes,macrophages, neutrophils, eosinophils, and basophils) or the lymphoidlineage (T and B lymphocytes and natural killer cells). The majorfunction of the myeloid lineage cells (e.g., neutrophils andmacrophages) is the phagocytosis of infectious organisms, live unwanteddamaged cells, senescent and dead cells (apoptotic and necrotic), aswell as the clearing of cellular debris. Phagocytes from healthy animalsdo not replicate and are diploid, i.e., have a DNA index of one. Onaverage, each cell contains <10 ng DNA, <20 ng RNA, and <300 ng ofprotein.

Distinct gene expression patterns of variation, e.g., those associatedwith cell type, gender, age, interindividual differences and the like,have been recognized in WBCs of healthy donors. For example, a“lymphocyte-associated” cluster has 55 unique genes. In neutrophils,significant variability in the expression of 52 unique gene clusters hasalso been reported. The genes in this cluster can be grouped into threeincreasingly specific families: (i) those ubiquitously expressed in manytypes of circulating immune cells; (ii) those expressed by cells of themyeloid lineage; and (iii) those specific to granulocytes.

The lifetime of various WBC subpopulations varies from a few days (e.g.,neutrophils) to several months (e.g., macrophages). Like other celltypes, leukocytes age and eventually die. During their aging process,human blood- and tissue-derived phagocytes (e.g., neutrophils) exhibitall the classic markers of programmed cell death (i.e., apoptosis),including caspase activation, pyknotic nuclei, and chromatinfragmentation. These cells also display a number of “eat-me” flags(e.g., phosphatidylserine, sugars) on the extracellular surfaces oftheir plasma membranes. Consequently, dying and dead cells andsubcellular fragments thereof are cleared from tissues and blood byother phagocytic cells.

The apoptosis of phagocytes is accelerated following their activation.For example, following the engulfment of S. aureus by neutrophils,phosphatidylserine is externalized on their plasma membranes, therebyleading to their rapid phagocytosis by macrophages. Activated monocyteshave also been shown to bind various tumor-cell lines with elevatedlevels of phosphatidylserine.

Circulating phagocytic cells are known to engulf live and dead CTCs andfragments thereof, a process that leads to an increase in the DNA (andother cellular constituent) contents of the phagocytosing cell. Forexample, apoptotic tumor cells have been shown to be phagocytosed bymacrophages and dendritic cells. Consequent to such phagocytic activity,blood macrophages obtained from prostate cancer patients have been shownto contain intracellularly much higher levels of prostate-specificantigen (PSA) than macrophages obtained from patients with benignprostate conditions. See, Herwig et al., Clinical Prostate Cancer 3, 184(2004) and Herwig et al., Prostate 62 290 (2005). This is believed to bea consequence of phagocytosing tumor cells. Fetal stem cells, nucleatederythrocytes, fetal lymphocytes, as well as significant amounts ofcell-free fetal nucleic acids are known to circulate in maternal blood.See Cheung et al., Nat. Genet. 14, 264 (1996).

It has also been shown that when apoptotic bodies (membrane-encapsulatedcell fragments) derived from human Burkitt's lymphoma cells are culturedwith human monocytes (phagocytic) or vascular smooth muscle cells(non-phagocytic), the monocytes show a high percentage of Epstein-Barrvirus (EBV)-specific, tumor-gene-positive cells, whereas smooth musclecells exhibit approximately 0.01% frequency of uptake and expression.

Methods are needed that enable the early diagnosis of the presence ofdisease (e.g., tumors) in individuals, e.g., individuals who are notknown to have the disease or who have recurrent disease. One object ofthe present invention is to facilitate the detection of disease-specific(e.g., tumor-specific) markers, e.g., proteins, RNA, DNA, carbohydratesand/or lipids and the like within subpopulations of white blood cells(WBCs) in an animal, including a human.

SUMMARY

Embodiments of the present invention are based on the use of phagocytesto determine the presence or absence of markers associated with certaindiseases or conditions. According to certain embodiments of the presentinvention, phagocytes incorporate cells and/or fragments and/orcomponents thereof circulating in blood that are characteristic of aparticular disease or condition. The contents of the phagocytes providea marker profile for the disease or condition, for example through DNAand/or proteins content in the cell or through DNA or protein expressionby the cell. Comparison of DNA expression profiles of phagocytic andnon-phagocytic WBC lead to the detection of tumor specific, diseasespecific or condition specific DNA signatures within phagocytic cellsthat were either not expressed or under-expressed in the non-phagocyticcell. Likewise, protein expression profiles of phagocytic andnon-phagocytic WBC lead to the detection of tumor specific, diseasespecific or condition specific protein signatures within phagocyticcells that were either not expressed or under-expressed in thenon-phagocytic cell. Accordingly, in certain embodiments, the methods ofthe present invention identify the presence of solid tumors (e.g.,primary and metastatic lesions) in an individual suspected of havingcancer and/or identify the presence of cancer prior to the manifestationof pathologic signs and symptoms and detect disease recurrence.According to other embodiments, the methods of the present inventiondiagnose certain diseases or other conditions by identifying specificsignatures from blood or other bodily fluid.

The present invention is based in part on the discovery that blood cellcomponents, such as phagocytic cells and non-phagocytic cells, of anindividual are ideally suited for the facile identification anddifferentiation of tumor specific and normal, non-specific signaturesand therefore the elimination of the inequality of baseline consequentto intrinsic interindividual (e.g., age, gender, ethnic background,health status) and temporal variations in gene expressions.

In certain exemplary embodiments, methods for the identification oftumor- and/or other disease-specific signatures within the WBCs(obtained from the blood or other bodily fluids, e.g., urine, stool,saliva, lymph, cerebrospinal fluid and the like) of an individualsuspected of having cancer and/or one or more other diseases ordisorders or conditions are provided. Embodiments of the presentinvention provide patient specific results and are not dependent onpopulation-derived average signature profiles and values obtained from“healthy” controls, i.e., the baseline/background signature(s) is/arespecific to the genomic, proteomic, metabolomic, glycomic,glycoproteomic, lipidomic, and/or lipoproteomic profile(s) of theindividual being evaluated. Embodiments of the present invention providea personalized predisposition to, screening, diagnosis, and monitoringof disease.

In certain embodiments and with reference to FIG. 1, the presentinvention is based on the ability of phagocytic cells to engulf andingest viable, dying and dead cells (e.g., apoptotic cells, necroticcells), microorganisms (e.g., bacteria (e.g., Rickettsia), viruses,fungi, yeast, protozoa and the like) subcellular particles and/orfragments thereof (cajal bodies, cell membrane, centrioles, centrosomes,gems, golgi apparatus, lysosomes, mitochondria, nuclear membrane,nuclei, nucleolus, paraspeckles, promyelocytic leukemia bodies (PMLbodies), ribosomes, rough endoplasmic reticulum, smooth endoplasmicreticulum, vacuoles, vesicles, microvesicles, and the like), andcellular debris, e.g. chromosomes, DNA (nuclear and mitochondrial),exons, genes, introns, proteins, prions, carbohydrate-binding proteins,glycoproteins, lipoproteins, RNA, microRNA, lipids, apoptotic bodies,nuclei, microvesicles, exosomes, nucleosomes, polymorphic interphasekaryosomal associations (PIKA), splicing spreckles, and the like), andthe absence of these characteristics in non-phagocytic cells.Accordingly, the analysis of DNA (nuclear, mitochondrial), RNA,microRNA, protein, prions, carbohydrate binding proteins, glycoproteins,lipids, lipoproteins, apoptotic bodies, nuclei, microvesicles, exosomesand/or nucleosomes and/or expression profiles of phagocytic WBCs andtheir comparison with those from non-phagocytic cells obtained from theblood or other bodily fluids of the same donor provides anidentification of tumor- and/or disease-specific signatures within thephagocytic cells (patient-specific signal) that are either not expressedor significantly differentially expressed in the non-phagocytic cells(patient-specific noise). Since both phagocytic and non-phagocytic cellsarise from the same pluripotent stem cell within the bone marrow,subtraction of the non-tumor-associated/induced signature profile(identified in the non-phagocytic cells) from the signatures found inthe phagocytic cells allows the identification of tumor- and/ordisease-specific signatures in the sample of the particular patient asshown in FIG. 2. According to certain other embodiments, cellular debrisin bodily fluids is internalized by entosis (cell absorption),endocytosis and pinocytosis.

According to one embodiment of the present invention and with referenceto FIG. 3, a blood sample is obtained from an individual with the bloodsample including both phagocytic and non-phagocytic cells (e.g., WBCs).Phagocytic cells(s) (e.g., neutrophils, monocytes, macrophages dendriticcells, foam cells) are then separated from non-phagocytic (e.g., Tcells, B cells, null cells, basophils) cell(s) by various methods knownto those of skill in the art. According to the present invention, thephenotype of WBCs is altered by the phagocytosis of live/dying/dead CTCs(and subcellular fragments thereof) and/or tumor- and/ordisease-specific DNA, RNA, protein, carbohydrate and/or lipid present inblood. Phagocytosis leads to the internalization of these tumor and/ordisease signatures into the phagocytosing cell and possibly theintegration of tumor-cell DNA with its tumor-specific somatic mutations(or other disease-related mutations) into the normal phagocytic cell DNA(i.e., its transfection of the chromosomes of the target cell). Thesubsequent transcription of the “transfected” DNA of phagocytic cellsinto RNA and the translation of the latter into proteins produces aphenotype different from non-phagocytic WBCs.

Therefore, comparison using genomic, proteomic, metabolomic, glycomic,glycoproteomic, lipidomic and/or lipoproteomic methods known to those ofskill in the art of the DNA, RNA, protein, and/or lipid expressionprofiles of phagocytic and non-phagocytic WBCs (as shown in FIG. 3)obtained from an individual with cancer (and/or one or more otherdiseases) is used to identify tumor-specific (and/or disease-specificand/or condition specific) signature(s) and/or profile(s) selectively inthe phagocytic cells which confirm the presence of occult tumor(s) (orother diseases or conditions) in the individual. According to thepresent invention, the subtraction of the DNA, RNA, protein,carbohydrate and/or lipid profiles of phagocytic cells fromnon-phagocytic cells provides a method for the identification (e.g.,after genomic, proteomic, metabolomic, glycomic, glycoproteomic,lipidomic and/or lipoproteomic analyses) of tumor-specific (and/ordisease-specific) signatures in a blood sample (and/or other biologicalsample) of a particular patient and signify the presence of occulttumor(s) and/or other disease as shown in FIG. 2.

In certain exemplary embodiments, phagocytic and non-phagocytic cells(e.g., obtained from the blood or one or more other biological samples(e.g., urine, stool, saliva, lymph, cerebrospinal fluid and the like),are separated. Since the phagocytosis of CTCs (and subcellular fragmentsthereof) by phagocytic WBC leads to the internalization of the tumorcells into the cytoplasm of phagocytic cells, the quantity of DNA, RNA,protein, carbohydrate and/or lipid within phagocytic cells will behigher than that of non-phagocytic cells. Therefore, comparison of thequantity and profile of these components between the phagocytic andnon-phagocytic cells is used as an indication of the presence of cancer.

In certain exemplary embodiments, a method for diagnosing the presenceof a cancer cell in an individual is provided. The method includes thesteps of obtaining a first expression profile from a blood phagocyticcell from an individual, obtaining a second expression profile from ablood non-phagocytic cell from the individual, comparing the first andsecond expression profiles, identifying differential expression of oneor more markers specific to the first expression profile, and relatingthe differential expression of the one or more markers specific to thefirst expression profile to the presence of a cancer cell in theindividual.

In certain exemplary embodiments, a method for identifying atumor-specific signature in an individual having cancer is provided. Themethod includes the steps of obtaining a first expression profile from ablood phagocytic cell from an individual having cancer, obtaining asecond expression profile from a blood non-phagocytic cell from theindividual having cancer, comparing the first and second expressionprofiles, identifying differential expression of two or more markersspecific to the first expression profile, and relating the differentialexpression of the two or more markers specific to a tumor-specificsignature in the individual having cancer.

In certain exemplary embodiments, a method for diagnosing the presenceof a cancer cell in an individual including the steps of obtaining afirst expression profile from a blood phagocytic cell from an individualand obtaining a second expression profile from a blood non-phagocyticcell from the individual is provided. The method includes the steps ofcomparing the first and second expression profiles, identifying thepresence of a circulating tumor cell or subcellular fragment thereofspecific to the first expression profile, and relating the presence of acirculating tumor cell or subcellular fragment thereof to the presenceof a cancer cell in the individual. In certain aspects, an increase inthe quantity of a marker in the first expression profile relative to thesecond expression profile indicates the presence of a circulating tumorcell or subcellular fragment thereof.

In certain exemplary embodiments and with reference to FIGS. 4-6, amethod for diagnosing the presence of a cancer cell in an individualincluding the steps of isolating a population of phagocytic cells froman individual and separating 2n phagocytic cells from >2n phagocyticcells is provided. The method includes the steps of obtaining a firstexpression profile from the 2n phagocytic cells, obtaining a secondexpression profile from the >2n phagocytic cells, comparing the firstand second expression profiles, and identifying differential expressionof one or more markers specific to the first expression profile. Themethod also includes the step of relating the differential expression ofthe one or more markers specific to the first expression profile to thepresence of a cancer cell in the individual.

In certain aspects of the methods described herein, markers include DNA,RNA, microRNA (e.g., DNA or RNA corresponding to cancer gene, oncogene,a tumor suppressor gene or any combination of these), protein (e.g., aprotein or polypeptide encoded by a cancer gene, oncogene, a tumorsuppressor gene or any combination of these), lipid, carbohydrate and/orany combination of these. In certain aspects, a blood phagocytic cell isa neutrophil, a macrophage, a monocyte, a dendritic cell, an eosinophil,a foam cell or any combination of these. In certain aspects, a bloodnon-phagocytic cell is a T cell, a B cell, a null cell, a basophil orany combination thereof. In other aspects, a blood phagocytic cell and ablood non-phagocytic cell are isolated from whole blood using methodsknown to those skilled in the art, such as antibodies. In still otheraspects, a blood phagocytic cell and a blood non-phagocytic cell areisolated from a population of white blood cells using methods know tothose of skill in the art such as fluorescence activated cell sorting(FACS). In other aspects, the blood phagocytic cell and the bloodnon-phagocytic cell are separated using a ligand that binds to amolecular receptor expressed on the plasma membranes of WBC populations.In yet other aspects, the blood phagocytic cell and the bloodnon-phagocytic cell are separated by one or methods includingfiltration, gradient-based centrifugation, elution, microfluidics andthe like. In certain aspects, an individual has one or more of occult(e.g., dormant, undiagnosed, hidden or concealed) cancer, previouslydiagnosed primary cancer and metastatic cancer. In certain aspects, amethod includes the step of relating the presence of one or more markersto efficacy of a cancer therapy.

In certain exemplary embodiments, the above described methods areapplied to detect, identify or diagnose the presence of an infectiousagent or disease other than cancer by comparing expression profiles ofphagocytic and nonphagocytic cells to determine differential expressionof markers characteristics of the infectious agent or disease other thancancer. In yet another aspect, one or more of the methods describedherein are used to detect the DNA, RNA, protein, carbohydrate and/orlipid profiles of pathogens (e.g., viruses, bacteria, rickettsia,protozoans, helminthes, fungi, yeasts and the like) and other diseasesor pathologies (e.g., Alzheimer's, dementia, heart failure,atherosclerosis, arthritis, genetic disorders, bone diseases,gastrointestinal diseases, prion diseases, and infectious diseases).

In certain aspects of the methods described herein, markers includepathogen DNA, pathogen RNA, pathogen protein, pathogen polypeptide,pathogen lipid and any combination of these. In certain aspects, aninfectious agent is a virus, a bacterium, a fungus, a parasite, aninfectious protein and any combination of these. In certain aspects, amethod includes the step of relating the presence of one or more markersto the efficacy of a pathogen therapy.

The methods and compositions described herein, therefore, enable thefacile identification of tumor specific signatures in the blood sampleof a patient, without depending on population-derived average signatureprofiles and values obtained from “healthy” controls. Specifically, themethods and compositions described herein can easily and economically:(i) identify tumor (primary and metastatic lesions) presence in anindividual prior to the manifestation of pathologic signs and symptoms;(ii) identify tumor (primary and metastatic lesions) presence in anindividual suspected of having cancer; and/or (iii) detect tumor(primary and metastatic lesions) recurrence in an individualundergoing/following various treatments.

Accordingly, the methods and compositions described herein (i) enablethe noninvasive screening of cancer; (ii) allow the diagnosis of tumors,especially at the earliest time points; (iii) move meaningfulintervention(s) to a much earlier point in the path of tumorprogression, thereby forestalling the development of metastatic disease;(iv) monitor the early response to routine or experimental treatment(s);(v) predict response to routine or experimental treatment(s); (vi)facilitate the selection of effective treatment by allowing rapididentification of ineffective treatments whose side effects might not bebalanced by expected benefits; (vii) minimize patient inconvenience andincapacitation; (viii) allow tumor detection, diagnosis, and treatmentto be closely coupled (e.g., personalization of anticancer therapy);(ix) provide for prediction and early detection of tumor type andstaging; (x) provide for therapy selection; (xi) determine whether atumor is metastatic or not; (xii) provide methods for the monitoring ofdiseases; and (xiii) methods for the prognosis of diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1 schematically depicts a proposed pathway leading to acquisitionof tumor-specific DNA, RNA, protein and/or lipid signatures byphagocytes following engulfment of live CTCs, apoptotic CTCs, fragmentedCTCs, tumor DNA, RNA, proteins, and lipids released by viable and/orapoptotic tumor cells. Note that only phagocytic cells (and notnon-phagocytic cells) acquire tumor signatures.

FIG. 2 schematically depicts an analytical method used in theidentification of cancer signatures expressed in/by phagocytic cells ofpatients with ovarian cancer (OC).

FIG. 3 schematically depicts a general flowchart of one embodiment of amethod of the invention.

FIG. 4 schematically depicts a proposed pathway leading to acquisitionof tumor-specific DNA, RNA, protein and lipid signatures by bloodphagocytes following engulfment of live CTCs, apoptotic CTCs, fragmentedCTCs, tumor DNA, RNA, proteins and lipids released by viable and/orapoptotic tumor cells. Note that DNA contents of phagocytes followingphagocytosis is >2n.

FIG. 5 schematically depicts analytical approaches used in theidentification of breast cancer (BC) signatures in BC-bearing animals.

FIG. 6 schematically depicts a general flowchart of another embodimentof a method of the invention.

FIG. 7 depicts gel electrophoresis analysis of total RNA isolated fromLNCaP and LLC1 cells.

FIG. 8 lists the yield and quality of RNA obtained from mouse whiteblood cells (WBCs).

FIGS. 9A-9D depict arrays showing seven up-regulated (≧2 fold), cancerrelated genes detected in neutrophils from LNCaP (human prostate cancer)tumor-bearing nude mice. (A) LNCaP tumor. (B) Neutrophils obtained fromnude mice bearing LNCaP tumors (N_(T)). (C) T cells obtained from nudemice bearing LNCaP tumors (T_(T)). (D) Neutrophils obtained fromnon-tumor-bearing nude mice (N_(N)). Circled signatures expressed intumor cells (A) and in neutrophils from tumor-bearing mice (B), andminimally expressed in neutrophils from non-tumor-bearing mice (D), andin non-phagocytic T cells (C). Expression in N_(T) was ≧2-fold than thatin N_(N) and T_(T).

FIGS. 10A-10D depict arrays showing three up-regulated, cancer relatedgenes detected in macrophages from LNCaP (human prostate cancer)tumor-bearing nude mice. (A) LNCaP tumor. (B) macrophages obtained fromnude mice bearing LNCaP tumors (M_(T)). (C) T cells obtained from nudemice bearing LNCaP tumors (T_(T)). (D) macrophages obtained fromnon-tumor-bearing nude mice (M_(N)). Circled signatures expressed intumor cells (A) and in macrophages from tumor-bearing mice (B), andminimally expressed in macrophages from non-tumor-bearing mice (D), andin non-phagocytic T cells (C). Expression in M_(T) was ≧2-fold than thatin M_(N) and T_(T).

FIGS. 11A-11D depict arrays showing four up-regulated (≧2 fold), cancerrelated genes detected in neutrophils from LS174T (human colon cancer)tumor-bearing nude mice. (A) LS174T tumor. (B) Neutrophils obtained fromnude mice bearing LS174T tumors (N_(T)). (C) T cells obtained from nudemice bearing LS174T tumors (T_(T)). (D) Neutrophils obtained fromnon-tumor-bearing nude mice (N_(N)). Circled signatures expressed intumor cells (A) and in neutrophils from tumor-bearing mice (B), andminimally expressed in neutrophils from non-tumor-bearing mice (D), andin non-phagocytic T cells (C). Expression was N_(T) is ≧2-fold than thatin N_(N) and T_(T).

FIGS. 12A-12D depict arrays showing three up-regulated (≧2 fold), cancerrelated genes detected in macrophages from LS174T (human colon cancer)tumor-bearing nude mice. (A) LS174T tumor. (B) Macrophages obtained fromnude mice bearing LS174T tumors (M_(T)). (C) T cells obtained from nudemice bearing LS174T tumors (T_(T)). (D) Macrophages obtained fromnon-tumor-bearing nude mice (M_(N)). Circled signatures expressed intumor cells (A) and in macrophages from tumor-bearing mice (B), andminimally expressed in macrophages from non-tumor-bearing mice (D), andin non-phagocytic T cells (C). Expression in M_(T) is ≧2-fold than thatin M_(N) and T_(T).

FIGS. 13A-13D depict arrays showing five up-regulated (≧2 fold), cancerrelated genes detected in neutrophils from LLC1 (mouse metastatic lungcancer) tumor-bearing C57/B1 mice. (A) LLC1 tumor. (B) Neutrophilsobtained from C57/B1 mice bearing LLC1 tumors (N_(T)). (C) T cellsobtained from C57/B1 mice bearing LLC1 tumors (T_(T)). (D) Neutrophilsobtained from non-tumor-bearing C57/B1 mice (N_(N)). Circled signaturesexpressed in tumor cells (A) and in neutrophils from tumor-bearing mice(B), and minimally expressed in neutrophils from non-tumor-bearing mice(D), and in non-phagocytic T cells (C). Expression in N_(T) was ≧2-foldthan that in N_(N) and T_(T).

FIGS. 14A-14D depict arrays showing two up-regulated (≧2 fold), cancerrelated genes detected in macrophages from LLC1 (mouse metastatic lungcancer) tumor-bearing C57/B1 mice. (A) LLC1 tumor. (B) Macrophagesobtained from C57/B1 mice bearing LLC1 tumors (M_(T)). (C) T cellsobtained from C57/B1 mice bearing LLC1 tumors (T_(T)). (D) Macrophagesobtained from non-tumor-bearing C57/B1 mice (M_(N)). Circled signaturesexpressed in tumor cells (A) and in neutrophils from tumor-bearing mice(B), and minimally expressed in neutrophils from non-tumor-bearing mice(D), and in non-phagocytic T cells (C). Expression in M_(T) was ≧2-foldthan that in M_(N) and T_(T).

FIG. 15A-15D depict arrays showing two up-regulated (≧2 fold), cancerrelated genes detected in neutrophils from B16F10 (mouse metastaticmelanoma) tumor bearing C57/B1 mice. (A) B16F10 tumor. (B) Neutrophilsobtained from C57/B1 mice bearing B16F10 tumors (N_(T)). (C) T cellsobtained from C57/B1 mice-bearing B16F10 tumors (T_(T)). (D) Neutrophilsobtained from non-tumor-bearing C57/B1 mice (N_(N)). Circled signaturesexpressed in tumor cells (A) and in neutrophils from tumor-bearing mice(B), and minimally expressed in neutrophils from non-tumor-bearing mice(D), and in non-phagocytic T cells (C). Expression in N_(T) was ≧2-foldthan that in N_(N) and T_(T).

FIG. 16A-16D depict arrays showing one up-regulated (≧2 fold), cancerrelated genes detected in macrophages from B16F10 (mouse metastaticmelanoma) tumor-bearing C57/B1 mice. (A) B16F10 tumor. (B) Macrophagesobtained from C57/B1 mice bearing B16F10 tumors (M_(T)). (C) T cellsobtained from C57/B1 mice bearing B16F10 tumors (T_(T)). (D: Macrophagesobtained from non-tumor-bearing C57/B1 mice (M_(N)). Circled signaturesexpressed in tumor cells (A) and in macrophages from tumor-bearing mice(B), and minimally expressed in macrophages from non-tumor-bearing mice(D), and in non-phagocytic T cells (C). Expression in M_(T) was ≧2-foldthan that in M_(N) and T_(T).

FIG. 17A-17D depict arrays showing five up-regulated (≧2 fold), cancerrelated genes detected in neutrophils from patient with head and neckcancer (squamous cell carcinoma). (A) Normal tissue (skin) biopsy. (B)Tumor tissue biopsy. (C) Neutrophils obtained from patient blood(N_(T)). (D) T cells obtained from patient blood (T_(T)). Circledsignatures expressed in tumor cells (B) and in neutrophils from patientblood (C), and minimally expressed or not expressed in normal skin (A)or non-phagocytic T cells (D). Expression in N_(T) was ≧2-fold than thatin T_(T) and skin.

FIG. 18A-18B depict arrays showing 23 up-regulated (≧2 fold), cancerrelated genes detected in macrophages from patient with ovarian cancer(adenocarcinoma). (A) Macrophages obtained from patient blood (M_(T)).(B) T cells obtained from patient blood (T_(T)). Circled signaturesexpressed in macrophages from patient (A) and minimally expressed innon-phagocytic T cells (B). Expression in M_(T) was ≧2-fold than that inT_(T).

FIG. 19 depicts a method used to identify tumor signatures in phagocyticcells. In this example, expression intensities of cancer associatedgenes in macrophages from tumor-bearing animals (M_(T)) were quantifiedcompared to those from T cells from the same animals (T_(T)) and thoseoverexpressed by >2-fold identified. Next, the intensities of allexpressed genes in M_(T) were quantified and compared to those inmacrophages obtained from non-tumor bearing animals (M_(NT)) and thegenes overexpressed >2-fold were identified. The genes common to bothlists were selected and compared to those expressed by the same tumor(shaded area).

FIGS. 20A-20D depict gene expression intensity comparisons in (A)macrophages obtained from nude mice bearing LNCaP human prostate tumors(M_(LNCaP)) and T cells from the same animals (Tcells_(LNCap)), (B)M_(LNCaP) and macrophages obtained from non-tumor-bearing mice(M_(non-tumor)), (C) neutrophils obtained from nude mice bearing LNCaPhuman prostate tumors (N_(LNCaP)) and T cells from the same animals(Tcells_(LNCaP)), and (D) N_(LNCaP) and macrophages obtained fromnon-tumor-bearing mice (N_(non-tumor)). Genes in red wereoverexpressed >2 fold; those in green were under-expressed >2 fold.

FIG. 21 lists expression of cancer-related genes within phagocyticneutrophils (N) and macrophages (M).

FIG. 22 lists cancer-related genes upregulated (>2-fold) in phagocyticmacrophages of a patient with ovarian cancer in comparison tonon-phagocytic T cells.

FIG. 23 depicts SDS gel (10%) electrophoresis of protein sample (5.9 g)obtained from mouse WBC.

FIG. 24 depicts Western blot analysis of TAG-72 and PSA expression in Tcells and monocytes/macrophages (M/M) obtained from tumor-bearing mice,illustrating the presence of signatures in phagocytic cells only.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Embodiments of the present invention are directed to a method ofproviding a patient-specific expression profile of markers associatedwith diseases, infectious agents and bodily conditions based on thecellular content and/or expression profiles of phagocytic cells.According to one aspect of the present invention, the cellular contentsand/or expression profiles of phagocytic cells is compared to knownmarkers for a particular disease state or condition to detect and/ordiagnose the particular disease state or condition. According to anadditional aspect of the present invention, the cellular content and/orexpression profile of phagocytic cells is compared to the cellularcontent and/or expression profile of non-phagocytic cells from the bloodof a single patient. Subtracting the cellular content and/or expressionprofile from non-phagocytic cells from that of phagocytic cells createsa cellular content and/or expression profile representative of only thedisease state of the individual.

According to an additional embodiment of the present invention, aphagocytic cell population from an individual is obtained and thecellular content and/or expression profile of phagocytic cells from thepopulation where the DNA content is greater than 2n is compared with thecellular content and/or expression profile of phagocytic cells from thesame population where the DNA content is 2n. According to a stilladditional embodiment of the present invention, a phagocytic cellpopulation from an individual is obtained and the expression profile ofphagocytic cells from the population where the RNA, protein,carbohydrate and/or lipid content is larger than normal and have a DNAindex greater than 1 is compared with the expression profile ofphagocytic cells from the same population where the RNA, protein,carbohydrate and/or lipid content is normal and/or have a DNA index of1.

Such a patient specific expression profile eliminates the dependence ona population-derived average signature profile for a particular diseaseor infectious agent, which may introduce error into the detection ordiagnosis of a particular disease in the individual. Such a patientspecific expression profile for a disease state of the present inventionallows detection, diagnosis and treatment to be personalized to theindividual.

With reference to FIGS. 1-3 and according to certain embodiments of thepresent invention, the gene expression profiles of phagocytic andnon-phagocytic WBCs obtained from mice bearing approximately three weekold human subcutaneous (s.c.) tumors (prostate LNCaP adenocarcinoma orLS174T colon adenocarcinoma) or mouse tumors (B16F10 metastaticmelanoma, administered intravenously, or LLC1 lung cancer, injecteds.c.), were compared. The results demonstrated that neutrophils andmacrophages obtained from these tumor-bearing mice express variousoncogenes and other cancer-related gene signatures that are alsoexpressed in each of the respective tumors. See FIGS. 9-16 and 19-21.These cancer-related genes and oncogenes (e.g., ERBB2, Jun, Fos, etc.)are not expressed or are minimally expressed by (i) non-phagocytic Tcells isolated from tumor-bearing mice, and (ii) neutrophils andmacrophages obtained from non-tumor-bearing mice. Furthermore, only thephagocytic cells from tumor-bearing mice were found to expresstumor-specific proteins. See FIGS. 23 and 24. CTCs and/or tumor-specificDNA and/or proteins in the blood of the mice were phagocytosed and someof the tumor-cell DNA, with its tumor-specific mutations and genes, wasintegrated, likely by transfection (without intending to be bound bytheory), into normal phagocyte DNA, transcribed into RNA, and translatedinto protein.

With reference to FIGS. 1-3 and according to certain exemplaryembodiments of the present invention, the gene expression profiles ofphagocytic and non-phagocytic WBCs obtained from patients with head andneck tumors or with ovarian cancer were also compared. The resultsdemonstrated that neutrophils and macrophages obtained from thesepatients express various oncogenes and other cancer-related genesignatures that are also expressed in each of the respective tumors. SeeFIGS. 17-18 and 22. These cancer-related genes and oncogenes were notexpressed or were minimally expressed by non-phagocytic T cells isolatedfrom the same individual patient. CTCs and/or tumor-specific DNA and RNAin the blood of the patient were phagocytosed and some of the tumor-cellDNA and/or RNA, with its tumor-specific mutations and genes, wasintegrated, likely through transfection (without intending to be boundby theory), into normal phagocyte DNA, transcribed into RNA, andtranslated into protein.

With reference to FIGS. 4-6 and according to certain exemplaryembodiments, the quantitative analysis of DNA (nuclear and/ormitochondrial), RNA, microRNA, protein, and/or lipid expression profilesof phagocytic cells (e.g., macrophages) obtained from the blood or oneor more other biological samples (e.g., urine, stool, saliva, lymph,cerebrospinal fluid and the like) whose (1) DNA content is >2n(P_(n>2)), or (2) RNA, protein, carbohydrate and/or lipid content islarger than normal, i.e., cells that have phagocytosed CTCs and/or theirsubcellular fragments or DNA/RNA/lipids (i.e., tumor-specific signaturesor other disease-specific signatures) and/or have a DNA index greaterthan one, and their comparison with the same phagocytic cell population(e.g., macrophages) whose (1) DNA content is 2n (P_(n=2)), or (2) RNA,protein, carbohydrate and/or lipid content is normal, i.e., cells thathave not phagocytosed CTCs and/or their subcellular fragments and have aDNA index of one, provides a method to detect tumor-specific (or otherdisease-specific) signatures within the P_(n>2) cells (patient-specificsignal) that are either not expressed or minimally expressed in theP_(n=2) cells (patient-specific noise). With reference to FIG. 6, thesubtraction of the DNA, RNA, protein, and/or lipid profiles of P_(n=2)from those of P_(n>2) as shown in FIG. 5 provides a method to identify(e.g., after one or more genomic, proteomic, metabolomic, glycomic,glycoproteomic, lipidomic and/or lipoproteomic analyses) tumor-specific(and/or disease-specific and/or condition specific) signatures in ablood sample (or one or more other biological samples such as, e.g.,other bodily fluids) of an animal and/or a human with cancer (and/ordisease and or bodily conditions) and signify the presence of occulttumor(s) and/or other disease and/or other conditions. Unlike themethods described above in which the genomic, proteomic, metabolomic,glycomic, glycoproteomic, lipidomic and/or lipoproteomic profiles ofphagocytic cells are compared with those of non-phagocytic cells, themajor advantages of this analytic detection method according to thepresent invention are: (i) it utilizes a single phagocytic cellsubpopulation as a source of the “tumor-specific” (e.g., P_(n>2)macrophage) and “normal-non-specific” (e.g., P_(n=2) macrophage)signatures, i.e., both share the same baseline genotype; and (ii) thesignature-acquiring cells (e.g., P_(n>2) neutrophil) are not dilutedwith those that have not phagocytosed, and therefore have not acquired,dead CTCs and/or fragments thereof (e.g., P_(n=2) neutrophils).

With reference to FIGS. 4-6 and according to certain exemplaryembodiments, the quantitative analysis of phagocytic cells (e.g.,macrophages) obtained from the blood or one or more other biologicalsamples or bodily fluid (e.g., urine, stool, saliva, lymph,cerebrospinal fluid and the like) whose intracellular content consequentto phagocytosis or internalization of other live, dying, or dead (e.g.,apoptotic or necrotic) cells, apoptotic bodies, nuclei, microvesicles,exosomes, nucleosomes, mitochondria, endoplasmic reticulum, and thelike, is greater than that of the same phagocytic cell population (e.g.,macrophages) with normal intracellular contents (P_(NIC)), i.e., cellsthat have not phagocytosed any of the above mentioned cells and/cellulardebris (patient-specific noise), provides a method to detecttumor-specific (or other disease-specific or other condition specific)signatures within the phagocytes with increased intracellular content(P_(IIC)) that are either not expressed or minimally expressed in thephagocytes with a normal intracellular contents (patient-specificnoise). With reference to FIG. 6, the subtraction of the DNA, RNA,protein, and/or lipid profiles of P_(NIC) from those of P_(IIC) as shownin FIG. 5 provides a method to identify (e.g., after one or moregenomic, proteomic, metabolomic, glycomic, glycoproteomic, lipidomic,and/or lipoproteomic analyses) tumor-specific (and/or disease-specific)signatures in a blood sample (or one or more other biological samplessuch as, e.g., other bodily fluids) of an animal with cancer or otherdisease and signify the presence of occult tumor(s) and/or otherdisease. Unlike the methods described above in which the genomic,proteomic, metabolomic, glycomic, glycoproteomic, lipidomic, and/orlipoproteomic profiles of phagocytic cells are compared with those ofnon-phagocytic cells, the major advantages of this analytic detectionmethod according to the present invention are: (i) it utilizes a singlephagocytic cell subpopulation as a source of the “disease-specific”(e.g., P_(IIC) macrophage) and “normal-non-specific” (e.g., P_(NIC)macrophage) signatures, i.e., both share the same baseline genotype; and(ii) the signature-acquiring cells (e.g., P_(IIC) neutrophil) are notdiluted with those that have not phagocytosed, and therefore have notacquired, dead CTCs and/or fragments thereof (e.g., P_(NIC)neutrophils).

The methods described herein (i) have high specificity, sensitivity, andaccuracy and should enable the detection of tumor-specific (and/or otherdisease-specific) and normal-nonspecific signatures present within ablood sample (or other biological sample such as, e.g., a bodily fluid);and (ii) eliminate the “inequality of baseline” that is known to occuramong individuals due to intrinsic (e.g., age, gender, ethnicbackground, health status and the like) and temporal variations in geneexpression. Accordingly, in certain aspects, the invention providesnon-invasive assays for the early detection of occult primary andmetastatic tumors (and/or one or more other diseases or conditions) inpatients, i.e., before the disease can be diagnosed by conventionalimaging techniques (e.g., PET, MRI, CT and the like), and, therefore,provide a foundation for improved decision-making relative to the needsand strategies for intervention, prevention, and treatment ofindividuals with cancer.

As used herein, the term “tumor specific marker” is intended to include,but is not limited to, one or more cellular components such as one ormore DNA sequences, one or more RNA sequences, one or more proteins, oneor more polypeptides, one or more lipids and the like. In certainaspects, a tumor specific marker is present in one or more WBCs such as,for example, a neutrophil, a macrophage and/or a dendritic cell.

As used herein, the term “cancer related genes” refers to genes such as,for example, cancer genes, oncogenes and/or tumor suppressor genes, thathave altered expression (e.g., increased expression or decreasedexpression when compared to a non-cancerous cell) in a cancerous cell(e.g., a WBC such as, for example, a macrophage, a neutrophil, a T cellor the like). Many cancer related genes are known in the art. Cancerrelated genes include for example, but are not limited to, ERBB2, JUN,RB1, SUPP1, MDM2, MAP2K1, MMP2, PDGFB, PLAUR, FGR, MYCL1, BLYM, NRAS1,PE1, SKI, TRK, ABL2, MYCN, RAB1, REL, RALB, LCO, ERBB4, RAF1, ECT2, KIT,FGF5, GRO1, GRO2, GRO3, FMS, PIM, KRAS1P, FYN, MYB, ROS1, MAS1, RALA,MYCLK1, GLI3, ARAF2, MET, BRAF, MOS, LYN, MYBL, MYC, OVC, VAV2, BMI1,RET, HRAS, SPI1, RELA, SEA, EMS1, ETS1, KRAS2, ERBB3, GLI, FLT, BRCA2,RB1, FOS, AKT1, ELK2, FES, MAF, TP53, CRK, ERBA1, NF1, EVI2, ERBBB2,INT4, BRCA1, YES1, JUND, JUNB, MEL, LPSA, VAV1, AKT2, FOSB, RRAS, HKR1,HKR2, ERBAL2, SRC, MYBL2, ETS2, ERG, ARAF1, YUASA, HS2, INT3, SNO, RMYC,BMYC, HRASP, TC21, TIM, PTI-1, JAK, one or members of the CEA family(see, e.g., Zhou et al. (2001) Gene 264:105), PSA, MUC-16 and the like.

As used herein, the term “cancer” refers to various types of malignantneoplasms, most of which can invade surrounding tissues, and maymetastasize to different sites (see, for example, PDR MedicalDictionary, 1st edition (1995), incorporated herein by reference in itsentirety for all purposes). The terms “neoplasm” and “tumor” refer to anabnormal tissue that grows by cellular proliferation more rapidly thannormal and continues to grow after the stimuli that initiatedproliferation is removed. Id. Such abnormal tissue shows partial orcomplete lack of structural organization and functional coordinationwith the normal tissue which may be either benign (i.e., benign tumor)or malignant (i.e., malignant tumor).

Examples of general categories of cancer include, but are not limitedto, carcinomas (i.e., malignant tumors derived from epithelial cellssuch as, for example, common forms of breast, prostate, lung and coloncancer), sarcomas (i.e., malignant tumors derived from connective tissueor mesenchymal cells), lymphomas (i.e., malignancies derived fromhematopoietic cells), leukemias (i.e., malignancies derived fromhematopoietic cells), germ cell tumors (i.e., tumors derived fromtotipotent cells. In adults most often found in the testicle or ovary;in fetuses, babies and young children, most often found on the bodymidline, particularly at the tip of the tailbone), blastic tumors (i.e.,a typically malignant tumor which resembles an immature or embryonictissue) and the like.

Examples of the types of neoplasms intended to be encompassed by thepresent invention include but are not limited to those neoplasmsassociated with cancers of neural tissue, blood forming tissue, breast,skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain,larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenalgland, immune system, head and neck, colon, stomach, bronchi, and/orkidneys.

In certain exemplary embodiments, one or more methods and/orcompositions described herein are applied to detect, identify and/ordiagnose disorders associated with the presence of fetal chromosomalabnormalities (e.g., Down's syndrome, autism and related autism spectrumdisorders (including, but not limited to, Asperger's syndrome andpervasive developmental disorder-not otherwise specified), sickle cellanemia, thalassemia and the like) consequent to the presence of fetalcells and DNA within maternal blood. Screening and diagnosing of one ormore of these disorders can be performed using the methods and/orcompositions described herein to detect one or more chromosomal markers,e.g., DNA and RNA, and the like, within maternal blood phagocytic cells.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be applied to test the gender of afetus within a pregnant woman by detecting the presence of fetus-derivedproteomic, lipidomic, and/or genomic signatures within blood of thepregnant woman, as fetal stem cells, nucleated erythrocytes, fetallymphocytes, as well as significant amounts of cell-free fetal nucleicacids are known to circulate in maternal blood. According to the methodsdescribed herein, the cellular content and/or expression profile ofphagocytic cells is compared to the cellular content and/or expressionprofile of non-phagocytic cells from the blood of a pregnant woman.Subtracting the cellular content and/or expression profile fromnon-phagocytic cells from that of phagocytic cells creates a cellularcontent and/or expression profile representative of the gender of thefetus being carried by the pregnant woman.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be used to detect, identify and/ordiagnose disorders associated with the presence of proteomic and/orgenomic myocyte signatures within blood of subjects having or at risk ofdeveloping cardiac disease (e.g., myocardial infarction, chronic heartfailure, ischemic heart disease, cardiovascular death and the like) bydetecting the presence of dying/dead myocytes and/or fragments thereof(e.g., DNA, proteins and the like). Screening and diagnosing of one ormore of these disorders is performed using methods and/or compositionsdescribed herein to detect one or more markers, e.g., DNA and RNA,protein and the like, within blood phagocytic cells.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be used to detect, identify and/ordiagnose disorders associated with the presence of proteomic, lipidomic,and/or genomic signatures within blood of subjects having or at risk ofdeveloping atherosclerosis consequent to coronary artery narrowing,abdominal aortic aneurism, and the like. Screening and diagnosing ofthese disorders can performed using the methods and/or compositionsdescribed herein to detect one or more markers, e.g., DNA, RNA, proteinand the like, within blood phagocytic cells.

Biopsy-confirmed rejection, one method for diagnosis of allograftrejection, is invasive and subject to sampling errors. Therefore, thedevelopment of noninvasive assays that detect molecular biomarkers fordiagnosing and managing transplanted organ rejection is useful inmanagement of transplant recipients by (a) detecting a pre-rejectionprofile that will allow therapeutic interventions before rejectioncauses graft dysfunction, (b) improving the sensitivity and specificityof rejection diagnosis, (c) developing new classification systems forrejection that will improve prognosis, and (d) providing information fordesigning individualized immunosuppressive regimens that could preventrejection while minimizing drug toxicity.

Accordingly, in certain exemplary embodiments, one or more methodsand/or compositions described herein can be used to detect, identify ordiagnose disorders associated with the presence of proteomic, lipidomic,and genomic signatures within blood of subjects having undergone organtransplants by detecting one or more markers, e.g., DNA, RNA, protein orthe like, within blood phagocytic cells.

Mitochondrial diseases result from failures of the mitochondria. Cellinjury and even cell death follow. Diseases of the mitochondria appearto cause the most damage to cells of the brain, heart, liver, skeletalmuscles, kidney and the endocrine and respiratory systems as well asdiabetes, respiratory complications, seizures, Alzheimer's disease,visual/hearing problems, lactic acidosis, developmental delays,susceptibility to infection, and cancer.

Accordingly, in certain exemplary embodiments, one or more methodsand/or compositions described herein can be used to screen, diagnoseand/or detect mitochondrial disease, by detecting one or more genomic,mitochondria-associated DNA markers within blood phagocytic cells.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be used to screen, diagnose and/ordetect Alzheimer's disease and/or dementia by detecting one or moremarkers, e.g., DNA, RNA, protein and the like, within blood phagocyticcells.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder thataffects various organs and systems. Accordingly, in certain exemplaryembodiments, one or more methods and/or compositions described hereincan be used to screen, diagnose and/or detect SLE by detecting one ormore markers, e.g., DNA, RNA, lipids, protein and the like, within bloodphagocytic cells.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be used to screen and/or detectgenomic and/or proteomic signatures useful in the development oftherapeutic and/or imaging molecules by detecting one or more markers,e.g., DNA, RNA, protein and the like, within blood phagocytic cells.

In certain exemplary embodiments, one or more methods and/orcompositions described herein can be used to screen, diagnose and/ordetect alteration in genomic, proteomic and/or lipidomic signaturesuseful in detection of diseases and pathologies consequent to one ormore external or internal insults (e.g., dirty bomb exposure, radiationexposure, chemical exposure, radiotherapy, radiopharmaceuticaladministration, therapeutic molecule exposure, radon exposure, asbestosexposure, pollution exposure and the like) by detecting one or moremarkers, e.g., DNA, RNA, protein, lipid and the like, within bloodphagocytic cells.

As used herein, the term “organism” includes, but is not limited to, ahuman individual, a non-human primate, a cow, a horse, a sheep, a goat,a pig, a dog, a cat, a rabbit, a mouse, a rat, a gerbil, a frog, a toadand a transgenic species thereof. The term “organism” further includespathogenic organisms, including, but not limited to, a pathogen such asa parasite, a yeast cell, a yeast tetrad, a yeast colony, a bacterium, abacterial colony, a virion, a virosome, a virus-like particle and/orcultures of any of these, and the like.

In certain exemplary embodiments, the assays described herein can beused for the detection of an infectious agent and/or the diagnosis of adisorder associated with an infection of a cell, tissue, organ or thelike by an infectious agent. In certain aspects, detection of aninfectious agent and/or the diagnosis of a disorder associated with aninfection is performed using the methods and/or compositions describedherein to detect one or more infectious agent markers, e.g., DNA, RNA,proteins, lipids and the like, from one or more infectious agents.

As used herein, the term “infectious agent” includes, but is not limitedto, pathogenic organisms such as viruses, bacteria, fungi, parasites,infectious proteins and the like.

Viruses include, but are not limited to, DNA or RNA animal viruses. Asused herein, RNA viruses include, but are not limited to, virus familiessuch as Picornaviridae (e.g., polioviruses), Reoviridae (e.g.,rotaviruses), Togaviridae (e.g., encephalitis viruses, yellow fevervirus, rubella virus), Orthomyxoviridae (e.g., influenza viruses),Paramyxoviridae (e.g., respiratory syncytial virus, measles virus, mumpsvirus, parainfluenza virus), Rhabdoviridae (e.g., rabies virus),Coronaviridae, Bunyaviridae, Flaviviridae, Filoviridae, Arenaviridae,Bunyaviridae and Retroviridae (e.g., human T cell lymphotropic viruses(HTLV), human immunodeficiency viruses (HIV)). As used herein, DNAviruses include, but are not limited to, virus families such asPapovaviridae (e.g., papilloma viruses), Adenoviridae (e.g.,adenovirus), Herpesviridae (e.g., herpes simplex viruses), andPoxviridae (e.g., variola viruses).

Bacteria include, but are not limited to, gram positive bacteria, gramnegative bacteria, acid-fast bacteria and the like.

As used herein, gram positive bacteria include, but are not limited to,Actinomedurae, Actinomyces israelii, Bacillus anthracis, Bacilluscereus, Clostridium botulinum, Clostridium difficile, Clostridiumperfringens, Clostridium tetani, Corynebacterium, Enterococcus faecalis,Listeria monocytogenes, Nocardia, Propionibacterium acnes,Staphylococcus aureus, Staphylococcus epiderm, Streptococcus mutans,Streptococcus pneumoniae and the like.

As used herein, gram negative bacteria include, but are not limited to,Afipia felis, Bacteroides, Bartonella bacilliformis, Bortadellapertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella,Calymmatobacterium granulomatis, Campylobacter, Escherichia coli,Francisella tularensis, Gardnerella vaginalis, Haemophilius aegyptius,Haemophilius ducreyi, Haemophilius influenziae, Heliobacter pylori,Legionella pneumophila, Leptospira interrogans, Neisseria meningitidia,Porphyromonas gingivalis, Providencia sturti, Pseudomonas aeruginosa,Salmonella enteridis, Salmonella typhi, Serratia marcescens, Shigellaboydii, Streptobacillus moniliformis, Streptococcus pyogenes, Treponemapallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis andthe like.

As used herein, acid-fast bacteria include, but are not limited to,Myobacterium avium, Myobacterium leprae, Myobacterium tuberculosis andthe like.

As used herein, other bacteria not falling into the other threecategories include, but are not limited to, Bartonella henseiae,Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasmapneumoniae, Rickettsia akari, Rickettsia prowazekii, Rickettsiarickettsii, Rickettsia tsutsugamushi, Rickettsia typhi, Ureaplasmaurealyticum, Diplococcus pneumoniae, Ehrlichia chafensis, Enterococcusfaecium, Meningococci and the like.

As used herein, fungi include, but are not limited to, Aspergilli,Candidae, Candida albicans, Coccidioides immitis, Cryptococci, andcombinations thereof.

As used herein, parasitic microbes include, but are not limited to,Balantidium coli, Cryptosporidium parvum, Cyclospora cayatanensis,Encephalitozoa, Entamoeba histolytica, Enterocytozoon bieneusi, Giardialamblia, Leishmaniae, Plasmodii, Toxoplasma gondii, Trypanosomae,trapezoidal amoeba and the like.

As used herein, parasites include worms (e.g., helminthes), particularlyparasitic worms including, but not limited to, Nematoda (roundworms,e.g., whipworms, hookworms, pinworms, ascarids, filarids and the like),Cestoda (e.g., tapeworms)

As used herein, infectious proteins include prions. Disorders caused byprions include, but are not limited to, human disorders such asCreutzfeldt-Jakob disease (CJD) (including, e.g., iatrogenicCreutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease(vCJD), familial Creutzfeldt-Jakob disease (fCJD), and sporadicCreutzfeldt-Jakob disease (sCJD)), Gerstmann-Strdussler-Scheinkersyndrome (GSS), fatal familial insomnia (fFI), sporadic fatal insomnia(sFI), kuru, and the like, as well as disorders in animals such asscrapie (sheep and goats), bovine spongiform encephalopathy (BSE)(cattle), transmissible mink encephalopathy (TME) (mink), chronicwasting disease (CWD) (elk, mule deer), feline spongiform encephalopathy(cats), exotic ungulate encephalopathy (EUE) (nyala, oryx, greaterkudu), spongiform encephalopathy of the ostrich and the like.

In certain exemplary embodiments, methods of detecting markers such asnucleic acid sequences (e.g., DNA, RNA and the like), proteins,polypeptides, lipids polysaccharides and the like in a biological sampleare provided. As used herein, the term “nucleic acid” is intended toinclude DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded.

As used herein, the term “amino acid” includes organic compoundscontaining both a basic amino group and an acidic carboxyl group.Included within this term are natural amino acids (e.g., L-amino acids),modified and unusual amino acids (e.g., D-amino acids and β-aminoacids), as well as amino acids which are known to occur biologically infree or combined form but usually do not occur in proteins. Naturalprotein occurring amino acids include alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tyrosine, tryptophan, proline, and valine. Naturalnon-protein amino acids include arginosuccinic acid, citrulline,cysteine sulfinic acid, 3,4-dihydroxyphenylalanine, homocysteine,homoserine, ornithine, 3-monoiodotyrosine, 3,5-diiodotryosine,3,5,5-triiodothyronine, and 3,3′,5,5′-tetraiodothyronine. Modified orunusual amino acids include D-amino acids, hydroxylysine,4-hydroxyproline, N-Cbz-protected amino acids, 2,4-diaminobutyric acid,homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine,phenylglycine, .alpha.-phenylproline, tert-leucine,4-aminocyclohexylalanine, N-methyl-norleucine, 3,4-dehydroproline,N,N-dimethylaminoglycine, N-methylaminoglycine,4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid,trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid,1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid.

As used herein, the term “peptide” includes compounds that consist oftwo or more amino acids that are linked by means of a peptide bond.Peptides may have a molecular weight of less than 10,000 Daltons, lessthan 5,000 Daltons, or less than 2,500 Daltons. The term “peptide” alsoincludes compounds containing both peptide and non-peptide components,such as pseudopeptide or peptidomimetic residues or other non-amino acidcomponents. Such compounds containing both peptide and non-peptidecomponents may also be referred to as a “peptide analog.”

As used herein, the term “protein” includes compounds that consist ofamino acids arranged in a linear chain and joined together by peptidebonds between the carboxyl and amino groups of adjacent amino acidresidues.

As used herein, the term “lipid” includes synthetic ornaturally-occurring compounds which are generally amphipathic andbiocompatible. Lipids typically comprise a hydrophilic component and ahydrophobic component. Exemplary lipids include, but are not limited tofatty acids, neutral fats, phosphatides, glycolipids and the like. Asused herein, the term “lipid composition” refers to a composition whichcomprises a lipid compound, typically in an aqueous medium. Exemplarylipid compositions include, but are not limited to, suspensions,emulsions, vesicle compositions and the like.

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample involves obtaining a biological sample (e.g., abodily fluid sample (e.g., blood) and/or tumor sample) from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting one or more markers (e.g., DNA, RNA, protein,polypeptide, carbohydrate, lipid or the like).

Detection methods described herein can be used to detect one or moremarkers (e.g., DNA, RNA, protein, polypeptide, carbohydrate, lipid orthe like) in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetection of a polypeptide corresponding to a marker of the inventioninclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of genomic DNA include Southern hybridizations. Furthermore,in vivo techniques for detection of a polypeptide corresponding to amarker of the invention include introducing into a subject a labeledantibody directed against the polypeptide. For example, the antibody canbe labeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

Methods for analyzing lipid content in a biological sample are known inthe art (See, e.g., Kang et al. (1992) Biochim. Biophys. Acta. 1128:267;Weylandt et al. (1996) Lipids 31:977; J. Schiller et al. (1999) Anal.Biochem. 267:46; Kang et al. (2001) Proc. Natl. Acad. Sci. USA 98:4050;Schiller et al. (2004) Prog. Lipid Res. 43:499). An exemplary method oflipid analysis is to extract lipids from a biological sample (e.g. usingchloroform:methanol (2:1, vol:vol) containing 0.005% butylatedhydroxytoluene (BHT, as an antioxidant)), prepare fatty acid methylesters were (e.g., 14% BF3-methanol reagent), and quantifying the fattyacid methyl esters are quantified (e.g., by HPLC, TLC, by gaschromatography-mass spectroscopy using commercially available gaschromatographs, mass spectrometers, and/or combination gaschromatograph/mass spectrometers). Fatty acid mass is determined bycomparing areas of various analyzed fatty acids to that of a fixedconcentration of internal standard.

A general principle of diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a marker (e.g.,one or more of DNA, RNA, protein, polypeptide, carbohydrate, lipid andthe like) and a probe under appropriate conditions and for a timesufficient to allow the marker and probe to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In certain exemplary embodiments, the probe, when it is the unanchoredassay component, can be labeled for the purpose of detection and readoutof the assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, U.S. Pat. Nos. 5,631,169 and 4,868,103). Afluorophore label on the first, ‘donor’ molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond ‘acceptor’ molecule, which in turn is able to fluoresce due tothe absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C., 1991, Anal. Chem. 63:2338 2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699 705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas and Minton (1993) TrendsBiochem. Sci. 18:284). Standard chromatographic techniques may also beutilized to separate complexed molecules from uncomplexed ones. Forexample, gel filtration chromatography separates molecules based onsize, and through the utilization of an appropriate gel filtration resinin a column format, for example, the relatively larger complex may beseparated from the relatively smaller uncomplexed components. Similarly,the relatively different charge properties of the marker/probe complexas compared to the uncomplexed components may be exploited todifferentiate the complex from uncomplexed components, for examplethrough the utilization of ion-exchange chromatography resins. Suchresins and chromatographic techniques are well known to one skilled inthe art (see, e.g., Heegaard (1998) J. Mol. Recognit. 11:141; Hage andTweed (1997) J. Chromatogr. B. Biomed. Sci. Appl. 12:499). Gelelectrophoresis may also be employed to separate complexed assaycomponents from unbound components (see, e.g., Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,1987 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, non-denaturinggel matrix materials and conditions in the absence of reducing agent aretypically preferred. Appropriate conditions to the particular assay andcomponents thereof will be well known to one skilled in the art.

In certain exemplary embodiments, the level of mRNA corresponding to themarker can be determined either by in situ and/or by in vitro formats ina biological sample using methods known in the art. Many expressiondetection methods use isolated RNA. For in vitro methods, any RNAisolation technique that does not select against the isolation of mRNAcan be utilized for the purification of RNA from blood cells (see, e.g.,Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley &Sons, New York 1987 1999). Additionally, large numbers of cells and/orsamples can readily be processed using techniques well known to those ofskill in the art, such as, for example, the single-step RNA isolationprocess of Chomczynski (1989, U.S. Pat. No. 4,843,155).

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. In certainexemplary embodiments, a diagnostic method for the detection of mRNAlevels involves contacting the isolated mRNA with a nucleic acidmolecule (probe) that can hybridize to the mRNA encoded by the genebeing detected. The nucleic acid probe can be, for example, afull-length cDNA, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to anmRNA or genomic DNA encoding a marker of the present invention. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein. Hybridization of an mRNA with the probe indicates thatthe marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in a gene chip array. A skilled artisan can readily adapt knownmRNA detection methods for use in detecting the level of mRNA encoded bythe markers of the present invention.

An alternative method for determining the level of mRNA corresponding toa marker of the present invention in a sample involves the process ofnucleic acid amplification, e.g., by rtPCR (the experimental embodimentset forth in U.S. Pat. Nos. 4,683,195 and 4,683,202), COLD-PCR (Li etal. (2008) Nat. Med. 14:579), ligase chain reaction (Barany, 1991, Proc.Natl. Acad. Sci. USA, 88:189), self sustained sequence replication(Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874),transcriptional amplification system (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86:1173), Q-Beta Replicase (Lizardi et al. (1988)Bio/Technology 6:1197), rolling circle replication (U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the sample(e.g., a bodily fluid (e.g., blood cells)) prior to detection. In suchmethods, a cell or tissue sample is prepared/processed using knownhistological methods. The sample is then immobilized on a support,typically a glass slide, and then contacted with a probe that canhybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in a patient sample from onesource to a patient sample from another source, e.g., to compare aphagocytic blood cell from an individual to a non-phagocytic blood cellfrom the individual.

In another exemplary embodiment, a protein or polypeptide correspondingto a marker is detected. In certain exemplary embodiments, an agent fordetecting a polypeptide of the invention is an antibody capable ofbinding to a polypeptide corresponding to a marker of the invention,such as an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled,”with respect to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

A variety of formats can be employed to determine whether a samplecontains a protein that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis, enzyme linkedimmunoabsorbant assay (ELISA) and the like. A skilled artisan canreadily adapt known protein/antibody detection methods for use indetermining whether cells (e.g., bodily fluid cells such as blood cells)express a marker of the present invention.

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, it is generally preferable toimmobilize either the antibody or proteins on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros,magnetite and the like.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated from cells(e.g., bodily fluid cells such as blood cells) can be run on apolyacrylamide gel electrophoresis and immobilized onto a solid phasesupport such as nitrocellulose. The support can then be washed withsuitable buffers followed by treatment with the detectably labeledantibody. The solid phase support can then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label on thesolid support can then be detected by conventional means.

In certain exemplary embodiments, diagnostic assays are provided. Anexemplary method for detecting the presence or absence of a bodilycondition, a disease and/or disorder associated with cancer, aninfectious agent, and/or another disease in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a compound or an agent capable of detecting oneor more of the markers of a disease and/or disorder associated withcancer, an infectious agent, and/or another disease or condition, e.g.,marker nucleic acid (e.g., mRNA, genomic DNA), marker peptide (e.g.,polypeptide or protein) or marker lipid encoded by the marker nucleicacid such that the presence of a marker nucleic acid or marker peptideencoded by the nucleic acid is detected in the biological sample. In oneembodiment, an agent for detecting marker mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to marker mRNA orgenomic DNA. The nucleic acid probe can be, for example, a full-lengthmarker nucleic acid or a portion thereof. Other suitable probes for usein the diagnostic assays of the invention are described herein.

An agent for detecting marker peptide can be an antibody capable ofbinding to a marker peptide, such as an antibody with a detectablelabel. Antibodies can be polyclonal or monoclonal. An intact antibody,or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. The term“labeled,” with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

As used herein, the term “biological sample” is intended to includetissues, cells (e.g., phagocytic cells, non-phagocytic cells, 2ncells, >2n cells and the like) and biological fluids (e.g., whole blood,WBCs and the like) isolated from a subject, as well as tissues, cells(e.g., phagocytic cells, non-phagocytic cells, 2n cells, >2n cells andthe like) and bodily fluids (e.g., urine, whole blood, WBCs and thelike) present within a subject. That is, the detection method of theinvention can be used to detect marker polypeptide, protein,carbohydrate, lipid, oligosaccharide, mRNA, microRNA, genomic DNA andthe like in a biological sample in vitro as well as in vivo. In oneembodiment, the biological sample contains proteins, polypeptides,lipids and/or oligosaccharides from the test subject. Alternatively, thebiological sample can contain mRNA molecules from the test subjectand/or genomic DNA molecules from the test subject. In one embodimentbiological sample is a serum sample, saliva sample or a biopsy sampleisolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject (e.g., non-phagocytic cell or2n cell), contacting the control sample (e.g., non-phagocytic cell or 2ncell) with a compound or agent capable of detecting marker polypeptide,protein lipid, oligosaccharide, mRNA, microRNA, genomic DNA and the likeis detected in the biological sample, and comparing the presence ofmarker polypeptide, protein lipid, oligosaccharide, mRNA, genomic DNAand the like in control sample with the presence of marker polypeptide,protein lipid, oligosaccharide, mRNA, genomic DNA and the like in thetest sample (e.g., phagocytic cell or >2n cell). Alternatively, thepresence of marker polypeptide, protein lipid, oligosaccharide, mRNA,genomic DNA and the like in the test sample (e.g., phagocytic cellor >2n cell) can be compared with information in a database or on achart to result in detection or diagnosis.

The invention also encompasses kits for detecting the presence of one ormore markers associated with cancer and/or an infectious agent in abiological sample. For example, the kit can comprise a labeled compoundor agent capable of detecting marker polypeptide, protein lipid,oligosaccharide, mRNA, microRNA, genomic DNA and the like in abiological sample; means for determining the amount of marker in thesample; and means for comparing the amount of marker in the sample witha standard (e.g., a non-phagocytic cell or a 2n cell). The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect marker peptide ornucleic acid.

In certain exemplary embodiments, prognostic assays are provided. Thediagnostic methods described herein can furthermore be utilized toidentify subjects having a condition or at risk of developing a diseaseand/or disorder associated with cancer and/or an infectious agent, oranother disorder described herein associated with upregulated (ordownregulated) expression of one or more of the markers describedherein. For example, the assays described herein, such as the precedingdiagnostic assays or the following assays, can be utilized to identify asubject having or at risk of developing a disease and/or disorderassociated with cancer and/or an infectious agent and/or one or moreother disorders described herein.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease and/or disorder associated withcancer and/or an infectious agent, and/or one or more other disordersdescribed herein associated with one or more of the markers describedherein. For example, such methods can be used to determine whether asubject can be effectively treated with an agent for treating,ameliorating or reducing one or more symptoms associated with cancer.Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disease and/ordisorder associated with cancer and/or an infectious agent, and/or oneor more other disorders described herein.

The methods of the invention can also be used to detect geneticalterations in a marker gene, thereby determining if a subject with thealtered gene is at risk for developing a disease and/or disorderassociated with cancer and/or an infectious agent, and/or one or moreother disorders described herein characterized by misregulation in amarker protein activity or nucleic acid expression, such as cancer. Incertain embodiments, the methods include detecting, in a sample of cells(e.g., bodily fluid cells such as blood cells) from the subject, thepresence or absence of a genetic alteration characterized by analteration affecting the integrity of a gene encoding a marker peptideand/or a marker gene. For example, such genetic alterations can bedetected by ascertaining the existence of at least one of: 1) a deletionof one or more nucleotides from one or more marker genes; 2) an additionof one or more nucleotides to one or more marker genes; 3) asubstitution of one or more nucleotides of one or more marker genes, 4)a chromosomal rearrangement of one or more marker genes; 5) analteration in the level of a messenger RNA transcript of one or moremarker genes; 6) aberrant modification of one or more marker genes, suchas of the methylation pattern of the genomic DNA; 7) the presence of anon-wild type splicing pattern of a messenger RNA transcript of one ormore marker genes; 8) a non-wild type level of a one or more markerproteins; 9) allelic loss of one or more marker genes; and 10)inappropriate post-translational modification of one or more markerproteins. As described herein, there are a large number of assays knownin the art which can be used for detecting alterations in one or moremarker genes.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195, 4,683,202 and 5,854,033), such as real-time PCR,COLD-PCR, anchor PCR, recursive PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)Science 241:1077; Prodromou and Pearl (1992) Protein Eng. 5:827; andNakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360), the latter ofwhich can be particularly useful for detecting point mutations in amarker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675). Thismethod can include the steps of collecting a sample of cells from asubject, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a marker gene under conditionssuch that hybridization and amplification of the marker gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA87:1874), transcriptional amplification system (Kwoh et al., (1989)Proc. Natl. Acad. Sci. USA 86:1173), Q-Beta Replicase (Lizardi et al.(1988) Bio-Technology 6:1197), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in one or more marker genes froma sample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,optionally amplified, digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared.

Differences in fragment length sizes between sample and control DNAindicates mutations in the sample DNA. Moreover, the use of sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations in one or more of the markersdescribed herein can be identified by hybridizing a sample and controlnucleic acids, e.g., DNA or RNA, to high density arrays containinghundreds or thousands of oligonucleotides probes (Cronin et al. (1996)Human Mutation 7: 244; Kozal et al. (1996) Nature Medicine 2:753). Forexample, genetic mutations in a marker nucleic acid can be identified intwo dimensional arrays containing light-generated DNA probes asdescribed in Cronin, M. T. et al. supra. Briefly, a first hybridizationarray of probes can be used to scan through long stretches of DNA in asample and control to identify base changes between the sequences bymaking linear arrays of sequential overlapping probes. This step allowsthe identification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence a marker gene anddetect mutations by comparing the sequence of the sample marker genewith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147).

Other methods for detecting mutations in a marker gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type marker sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to base pair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286. In one embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in marker cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657).According to an exemplary embodiment, a probe based on a markersequence, e.g., a wild-type marker sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in marker genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton(1993) Mutat. Res. 285:125; and Hayashi (1992) Genet. Anal. Tech. Appl.9:73). Single-stranded DNA fragments of sample and control markernucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In one embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucl. Acids Res. 17:2437) or at the extreme 3′ end of one primerwhere, under appropriate conditions, mismatch can prevent, or reducepolymerase extension (Prossner (1993) Tibtech 11:238). In addition itmay be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection (Gasparini et al. (1992)Mol. Cell Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, andaccompanying claims.

Example 1 Representative Method I for the Separation of Phagocytic Cellsfrom Non-Phagocytic Cells and the Analysis of Expression Profiles

1. With reference to FIGS. 2 and 3, coat plates with avidin.

2. Add biotinylated antibody to non-phagocytic blood cell (e.g., Tcells) to the wells, incubate for 30 min at RT, wash wells.

3. Add magnetic beads.

4. Add WBC blood sample.

5. Incubate at 37° C. (30 minutes-1 hour).

6. Following phagocytosis of beads by phagocytic cells and binding ofavidin-biotin-antibody to non-phagocytic cells, place plate on top ofmagnet and wash (the phagocytic cells that internalized the magneticbeads and the non-phagocytic cells bound to the antibody will stay; allother cells will be washed away).

7. Remove magnet and collect phagocytic cells.

8. Isolate RNA from phagocytic cells (e.g., cells bound to a magneticbead) and of non-phagocytic cells (e.g., those cells attached to thebottom of the wells via the anti-non-phagocytic cell biotinylatedantibody-avidin bound), prepare cDNA, cRNA and use to differentiategenetic profiles (e.g., whole gene arrays and/or cancer gene arrays) ofphagocytic and non-phagocytic cells.

9. Isolate DNA from each cell sample and identify tumor-DNA signaturesselectively present in phagocytes (i.e., absent in non-phagocytes);compare the profiles (e.g., whole gene arrays, DNA mutations and/or SNPsobtained in phagocytic and non-phagocytic cells).

10. Isolate protein from each cell sample, run Western blots usingantibodies to known proteins overexpressed by human tumors (e.g., PSAand PSMA in prostate cancer; CEA in colon cancer; and CA125 in ovariancancer), and compare the profiles obtained in phagocytic andnon-phagocytic cells. Alternatively, use mass spectroscopy to identifythe proteins.

11. Isolate lipids from each cell sample and compare quantity andquality, for example using HPLC.

Example 2 Representative Method II for the Separation of PhagocyticCells from Non-Phagocytic Cells and the Analysis of Expression Profiles

1. With reference to FIGS. 2 and 3, lyse RBCs in blood sample.

2. Cytospin WBC on glass slides.

3. Fix cells in acetone/methanol (−20° C. for 5 minutes).

4. Stain with hematoxylin and eosin stain and anti-T cell antibody.

5. Isolate T cells (non-phagocytic) and macrophages (phagocytic) usinglaser capture microscopy (LCM).

6. Isolate RNA from phagocytic cells and of non-phagocytic cells,prepare cDNA, cRNA and use to differentiate genetic profiles (e.g.,whole gene arrays and/or cancer gene arrays) of phagocytic andnon-phagocytic cells.

7. Isolate DNA from each cell sample, run DNA arrays, and compare theprofiles (e.g., whole gene arrays, DNA mutations and/or SNPs) obtainedin phagocytic and non-phagocytic cells.

8. Isolate protein from each cell sample, run Western blots usingantibodies to known proteins overexpressed by human tumors (e.g., PSAand PSMA in prostate cancer; CEA in colon cancer; and CA125 in ovariancancer), and compare the profiles obtained in phagocytic andnon-phagocytic cells. Alternatively, use mass spectroscopy to identifythe proteins.

9. Isolate lipids from each cell sample and compare quantity andquality, for example using HPLC.

Example 3 Representative Method III for the Separation of PhagocyticCells from Non-Phagocytic Cells and the Analysis of Expression Profiles

1. With reference to FIGS. 2 and 3, lyse RBC from a blood sample.

2. Use magnetic antibody-conjugated beads to isolate non-phagocytic(e.g., T cells) and phagocytic cells (e.g., neutrophils and/ormacrophages and/or monocytes) from whole blood.

3. Isolate RNA from each cell sample, prepare cDNA, cRNA and use todifferentiate genetic profiles (e.g., cancer gene array) of phagocyticand non-phagocytic cells.

4. Isolate DNA from each cell sample, run DNA arrays, and compare theprofiles obtained in phagocytic and non-phagocytic cells.

5. Isolate protein from each cell sample, run Western blots usingantibodies to known proteins overexpressed by human tumors (e.g., PSAand PSMA in prostate cancer; CEA in colon cancer; and CA125 in ovariancancer), and compare the profiles obtained in phagocytic andnon-phagocytic cells. Alternatively, use mass spectroscopy to identifythe proteins.

6. Isolate lipids from each cell sample and compare quantity andquality, for example using HPLC.

Example 4 Representative Method IV for the Separation of PhagocyticCells from Non-Phagocytic Cells and the Analysis of Expression Profiles

1. With reference to FIGS. 2 and 3, stain WBC with fluorescentantibodies specific against a particular cell subpopulation (e.g.,neutrophils, macrophages, monocytes, T cells and the like).

2. Sort the cells (e.g., by FACS).

3. Isolate RNA from each cell sample, prepare cDNA, cRNA and use todifferentiate genetic profiles (e.g., cancer gene array) of phagocyticand non-phagocytic cells.

4. Isolate DNA from each cell sample, run DNA arrays, and compare theprofiles obtained in phagocytic and non-phagocytic cells.

5. Isolate protein from each cell sample, run Western blots usingantibodies to known proteins overexpressed by human tumors (e.g., PSAand PSMA in prostate cancer; CEA in colon cancer; and CA125 in ovariancancer), and compare the profiles obtained in phagocytic andnon-phagocytic cells. Alternatively, use mass spectroscopy to identifythe proteins.

6. Isolate lipids from each cell sample and compare quantity andquality, for example using HPLC.

Example 5 Representative Method V for the Separation of Phagocytic Cellsfrom Non-Phagocytic Cells and the Analysis of Expression Profiles

1. With reference to FIGS. 5 and 6, stain WBC with fluorescentantibodies to each cell subpopulation (e.g., neutrophils, macrophages,monocytes, and T cells), and then stain with DNA dye (e.g., propidiumiodide).

2. Sort the cells (FACS) into T cells, neutrophils (2n), neutrophils(>2n), macrophages (2n), macrophages (>2n), monocytes (2n), andmonocytes (>2n).

3. Isolate RNA from T cells, neutrophils (>2n), macrophages (>2n), andmonocytes (>2n). Then prepare cDNA, cRNA and use to differentiategenetic profiles (e.g., cancer gene array) of phagocytic andnon-phagocytic cells.

4. Isolate DNA from T cells, neutrophils (>2n), macrophages (>2n), andmonocytes (>2n). Run DNA arrays and compare the profiles obtained inphagocytic and non-phagocytic cells.

5. Isolate protein from T cells, neutrophils (>2n), macrophages (>2n),and monocytes (>2n). Run Western blots using antibodies to knownproteins overexpressed by human tumors (e.g., PSA and PSMA in prostatecancer; CEA in colon cancer; and CA125 in ovarian cancer), and comparethe profiles obtained in phagocytic and non-phagocytic cells.Alternatively, use mass spectroscopy to identify the proteins.

6. Isolate lipids from T cells, neutrophils (>2n), macrophages (>2n),and monocytes (>2n).

Compare quantity and quality of lipids, for example using HPLC

Example 6 Representative Method VI for the Separation of PhagocyticCells and the Analysis of Expression Profiles

1. With reference to FIGS. 5 and 6, stain WBC with fluorescentantibodies specific against one or more phagocytic cells (e.g.,neutrophils, macrophages, or monocytes) and then stain with DNA-bindingdye (e.g., propidium iodide).

2. Sort the cells (FACS) into 2n and >2n phagocytes.

3. Isolate RNA from each of the 2n and >2n phagocytes. Prepare cDNA,cRNA and use to differentiate genetic profiles (e.g., cancer gene array)of 2n-phagocytic and >2n-phagocytic cells.

4. Isolate DNA from each of the 2n and >2n phagocytes. Run DNA arraysand compare the profiles obtained from 2n-phagocytic and >2n-phagocyticcells.

5. Isolate protein from each of the 2n and >2n phagocytes. Run Westernblots using antibodies to known proteins overexpressed by human tumors(e.g., PSA and PSMA in prostate cancer; CEA in colon cancer; and CA125in ovarian cancer), and compare the profiles obtained from 2n-phagocyticand >2n-phagocytic cells.

6. Isolate lipids from each of the 2n and >2n phagocytes. Comparequantity and quality of lipids, for example using HPLC.

Example 7 Detection of Tumor-Specific Gene Signatures in PhagocytesObtained from Tumor-Bearing Mice

According to embodiments of the present invention, methods are providedto differentiate between “normal non-specific noise” and“tumor-specific” and/or “disease-specific” signatures in blood or otherbodily fluids. The gene-expression profiles of bloodmonocytes/macrophages and neutrophils from tumor-bearing mice werecompared with that of non-phagocytic T cells from the same donor mice toidentify tumor-specific signatures within the phagocytic cells that wereeither not expressed or significantly differentially expressed innon-phagocytic cells from the same tumor-bearing mice and fromnon-tumor-bearing animals.

Human Prostate LNCaP Cancer Cells

Athymic nude mice (n=5) were injected subcutaneously (s.c.) with humanprostate LNCaP cancer cells. Twenty-seven days later (tumor size=˜0.4cm), the mice were bled by cardiac puncture (˜1 mL/mouse) intoEDTA-containing tubes that were then centrifuged. The buffy coat wasisolated and washed, and neutrophils, macrophages, and T cells wereseparated using, respectively, anti-mouse neutrophil-, macrophage-, andT cell-immunomagnetic DynaBeads. RNA was isolated from each cell sample(Triazol®). The RNA quality was determined as shown in FIG. 3. The RNAyield is shown in FIG. 20. cDNA and biotinylated cRNA (cRNA-B) wereprepared. Finally, the cRNA-B samples were incubated with cancer-genehuman microarrays (Oligo GEArray® Human Cancer PathwayFinderMicroarray—OHS-033—SuperArray Bioscience). Following hybridization, themembranes were washed and stained with avidin-alkaline phosphatase, andthe genes were detected using chemiluminescence (X-ray film).

Human LS174T Colon Adenocarcinoma Tumors, LLC1 Carcinoma Cells, B16F10Mouse Melanoma Cells

Similar experiments were carried out with cells isolated from athymicnude mice (n=5) injected s.c. with human LS174T colon adenocarcinomatumors (tumor size=˜0.3 cm), C57B1 mice (n=5) injected s.c. with Lewislung mouse LLC1 carcinoma cells (tumor size=˜0.6 cm), and C57B1 mice(n=5) injected intravenously 22 days earlier with 10⁶ B16F10 mousemelanoma cells (when the tumor cells were of mouse origin, the cRNA-Bsamples were hybridized with the Oligo GEArray® Mouse CancerPathwayFinder Microarray—OMM-033—SuperArray Bioscience). RNA was alsoisolated from exponentially growing LS174T, LLC1, B16F10, and LNCaPcells in culture and from neutrophils, macrophages and T cells isolatedfrom non-tumor-bearing C57B1 and nude mice, and their cancer-relatedgene profiles were determined.

According to the data obtained from these experiments and shown in FIGS.9-17, neutrophils and macrophages—obtained from mice injected with humanprostate or colon tumor cells and from mice bearing mouse lung cancer ormelanoma—have various cancer-related gene signatures that are also foundin their respective tumor cells. These cancer-related genes were notexpressed or were minimally expressed by (i) non-phagocytic T cellsisolated from tumor-bearing mice; and (ii) phagocytic neutrophils andmacrophages obtained from non-tumor-bearing mice.

For example, neutrophils isolated from the blood of nude mice bearingLNCaP human prostate cancer cells expressed several human tumor genesignatures (Human Cancer PathwayFinder Microarray) that were alsoexpressed in LNCaP cells (compare profiles of arrays in FIGS. 9A and9B). These genes were either not expressed or minimally expressed in Tcells obtained from tumor-bearing mice or neutrophils isolated fromnormal mice (see profiles in FIGS. 9C and 9D). Similarly, neutrophilsisolated from the blood of mice bearing LLC1 mouse lung cancer cellsexpressed several mouse tumor gene signatures (Mouse CancerPathwayFinder Microarray) that were expressed in LLC1 cells (compareprofiles of arrays in FIGS. 13A and 13B).

These genes were either not expressed or minimally expressed in T cellsobtained from tumor-bearing mice or neutrophils isolated from normalmice (see profiles shown in FIGS. 13C and 13D). Finally, the arrays werescanned, the intensity of each gene quantified using the softwareprovided by the company, and those genes overexpressed selectively byphagocytic cells identified as shown in FIGS. 19 and 20. FIGS. 21 and 22list the gene signatures acquired and differentially exhibited by thephagocytic WBCs of tumor-bearing mice. As shown in FIG. 21, manyoncogenes (genes depicted in red, e.g., ERBB2 and Jun) were detected andoften they were expressed simultaneously in macrophages and neutrophils.C57BI mice (n=5) were injected subcutaneously with 1E6 Lewis lung mousecarcinoma cells (LLCI). Twenty days later, the mice were anesthetizedand bled by cardiac puncture (approximately 1 mL/mouse) into anEDTA-containing tube. Following centrifugation at 2,000 rpm for 5minutes at room temperature, the buffy coat was transferred to a tubeand washed with PBS.

Anti-mouse macrophage/monocyte rat IgG antibodies (monocyte/macrophagemarker—F4/80—IgG2b from AbD Serotec, Raleigh, N.C.) were incubated (roomtemperature for 30 minutes) with anti-rat IgG antibody magnetic beads(DYNABEAD® sheep anti-rat IgG from INVITROGEN™, Carlsbad, Calif.). Theanti-macrophage/monocyte beads were then washed in PBS and stored onice.

Anti-mouse neutrophil rat IgG (Neutrophil Marker NIMP-R14—IgG2a—SantaCruz Biotechnology, Santa Cruz, Calif.) was incubated (room temperaturefor 30 minutes) with anti-rat IgG antibody magnetic beads (DYNABEAD®sheep anti-rat IgG—INVITROGEN™), washed in PBS, and stored on ice.

DYNABEAD® mouse Pan T (Thy1.2) beads (INVITROGEN™) were also washed inPBS and stored on ice.

Mouse blood macrophages and monocytes were isolated from the WBCsuspension prepared above using the anti-macrophage/monocyte beads. Inessence, the beads were added to the WBC sample and following theirincubation (4° C. for 30 minutes), the macrophage-bound beads wereisolated using a magnet and washed with PBS three times and stored onice.

Mouse T cells were then isolated from the remaining WBC. Briefly, theanti-mouse T cell beads were added to the WBC suspension, the samplesincubated (4° C. for 30 minutes), the T cell-bound beads were isolatedusing a magnet, washed with PBS, and stored on ice.

Finally, mouse neutrophils were isolated from the remaining WBC sample.The anti-mouse neutrophil magnetic beads were added to the cells and thesamples were incubated (4° C. for 30 minutes). The neutrophil-boundbeads were isolated using a magnet, washed with PBS, and stored on ice.

RNA was then isolated from each sample (using TRIZOL®, INVITROGEN™,Carlsbad, Calif.). The RNA quality was determined as shown in FIG. 7.The RNA yield is shown in FIG. 8. Next, cDNA (biotinylated) wereprepared and incubated (60° C. overnight) with cancer-gene humanmicroarrays (OLIGO GEARRAY® Human Cancer PathwayFinder MicroarrayOMM-033, SuperArray Bioscience, Frederick, Md.). Followinghybridization, the membranes were washed and stained withavidin-alkaline phosphatase and the genes detected usingchemiluminescence (X-ray film).

Human LS175T Colon Adenocarcinoma Tumors, LLC1 Carcinoma and B16F10Mouse Melanoma Cells

Similar experiments were carried out with cells isolated from athymicnude mice (n=5) injected s.c. with human LS174T colon adenocarcinomatumors (tumor size=˜0.3 cm), C57/B1 mice (n=5) injected s.c. with Lewislung mouse LLC1 carcinoma cells (tumor size=˜0.6 cm), and C57B1 mice(n=5) injected intravenously 22 days earlier with 10⁶ B16F10 mousemelanoma cells (when the tumor cells were of mouse origin, the cRNA-Bsamples were hybridized with the Oligo GEARRAY® Mouse CancerPathwayFinder Microarray—OMM-033—SuperArray Bioscience (when the tumorswere of human origin, the Oligo GEArray® Human Cancer PathwayFinderMicroarray—OHS-033—was used). RNA was also isolated from exponentiallygrowing LS174T, LLC1, B16F10, and LNCaP cells in culture and fromneutrophils, macrophages and T cells isolated from non-tumor-bearingC57B1 and nude mice, and their cancer-related gene profiles weredetermined.

According to the data obtained from these experiments and shown in FIGS.9A-9D, 10A-10D, 11A-11D, 12A-12D, 13A-13D, 14A-14D, 15A-15D, 16A-16D,neutrophils and macrophages (obtained from mice injected with humanprostate or colon tumor cells and from mice bearing mouse lung cancer ormelanoma) had various cancer-related gene signatures that were alsofound in their respective tumor cells (FIG. 21). These cancer-relatedgenes were not expressed or were minimally expressed by (i)non-phagocytic T cells isolated from tumor-bearing mice; and (ii)phagocytic neutrophils and macrophages obtained from non-tumor-bearingmice.

For example, neutrophils isolated from the blood of nude mice bearingLNCaP human prostate cancer cells expressed seven human tumor genesignatures (Human Cancer PathwayFinder Microarray) that were alsoexpressed in LNCaP cells (compare profiles of arrays in FIGS. 9A and9B). These genes were either not expressed or minimally expressed in Tcells obtained from tumor-bearing mice or neutrophils isolated fromnormal mice (see profiles in FIGS. 9C and 9D). Finally, the arrays werescanned, the intensity of each gene quantified using the softwareprovided by the company, and those genes overexpressed selectively byphagocytic cells identified as shown in FIGS. 19 and 20. FIGS. 21 and 22list the gene signatures acquired and differentially exhibited by thephagocytic WBCs of tumor-bearing mice. As shown in FIG. 21, manyoncogenes (e.g., ERBB2 and Jun) were detected and often they wereexpressed simultaneously in macrophages and neutrophils (shown by thegenes highlighted in green).

Example 8 Detection of Tumor-Specific Gene Signatures in PhagocytesObtained from Cancer Patients

According to certain embodiments of the present invention, thegene-expression profiles of blood monocytes/macrophages and neutrophilsfrom cancer patients were compared with that of non-phagocytic T cellsfrom the same donor individuals to identify tumor-specific signatureswithin the phagocytic cells that were either not expressed orsignificantly differentially expressed in non-phagocytic cells.

Patients with Head and Neck Tumors

Ten milliliters of venous blood was obtained (into an EDTA-containingtube) from patients known to have squamous cell carcinoma of the neckand scheduled for surgery. Following centrifugation at 2,000 rpm for 5minutes at room temperature, the buffy coat was transferred to a tubeand washed with PBS.

The cells were separated employing T cell-, neutrophil-, andmacrophage/monocyte-rat anti-human immunomagnetic DynaBeads® fromINVITROGEN™, Carlsbad, Calif. In essence, the beads were addedconsecutively to the WBC sample and following individual 4° C., 30minute incubations, the T cells-, neutrophils-, andmacrophages/monocytes-bound beads were isolated using a magnet andwashed with PBS three times.

RNA was then isolated from each sample (using TRIZOL®, INVITROGEN™,Carlsbad, Calif.). The RNA quantity and quality was determined and cDNAand biotinylated cRNA (cRNA-B) were prepared. Finally, the cRNA-Bsamples were incubated (60° C. overnight) with cancer-gene humanmicroarrays (Oligo GEArray® Human Cancer PathwayFinderMicroarray—OHS-033—SuperArray Bioscience, Frederick, Md.). Followinghybridization, the membranes were washed and stained withavidin-alkaline phosphatase, and the genes were detected usingchemiluminescence (X-ray film).

According to the data obtained from these experiments, neutrophils andmacrophages (obtained from head and neck cancer patients) had variouscancer-related gene signatures that were also found in their respectivetumor cells. These cancer-related genes were not expressed or wereminimally expressed by non-phagocytic T cells.

For example, neutrophils isolated from the blood of one such patientexpressed four human tumor gene signatures (Human Cancer PathwayFinderMicroarray) that were also expressed in the tumor biopsy obtained fromthe same patient (compare profiles of arrays in FIGS. 17B and 17C).These genes were either not expressed or minimally expressed in normalskin biopsy and in T cells isolated from the same blood sample (seeprofiles in FIGS. 17A and 17D, respectively). Finally, the arrays werescanned, the intensity of each gene quantified using the softwareprovided by the company, and the following genes that were overexpressed(>2-fold) selectively by phagocytic cells were identified: E26 viraloncogene homolog (ETS2), HIV-1 Tat interactive protein (HTAT1P2), IL8(neutrophil activation and chemotaxis), Jun oncogene (JUN), and matrixmetalloproteinase 9 (MMP9).

Ovarian Cancer Patients

Similar experiments were carried out with cells isolated from a patientwith ovarian cancer. According to the data obtained from theseexperiments, neutrophils and macrophages (obtained from the diseasedwoman) expressed many cancer-related genes that were not expressed orwere minimally expressed by non-phagocytic T cells.

For example, macrophages isolated from the blood of the ovarian cancerpatient expressed 23 human tumor gene signatures (Human CancerPathwayFinder Microarray) that were either not expressed or minimallyexpressed in T cells isolated from the same blood sample (compareprofiles in FIGS. 18A and 18B). Finally, the arrays were scanned, theintensity of each gene quantified using the software provided by thecompany, and the intensities of each cancer-related gene in each celltype determined. The list of 23 cancer-related genes differentiallyupregulated/overexpressed in macrophages as well as the macrophage-to-Tcell intensity ratios are both shown in FIG. 22. Note that a total offive oncogenes were detected (shown in red in FIG. 21).

Example 9 Detection of Tumor-Specific Protein Signatures in PhagocytesObtained from Mice Bearing Human Prostate LNCaP Tumors and Human ColonLS174T Tumors

A protein purification kit (Norgen, Incorporated, Product #23500) wasused to isolate and purify proteins from mouse WBCs, T cells, andmacrophages. The assay was very simple and fast (approximately 30minutes) and the isolated proteins, which were of high quality andexcellent yield (117.6±10.60 μg per 4 mL blood, n=5), could be used in anumber of downstream applications, such as SDS-PAGE analysis as shown inFIG. 23 and Western blots.

Protein samples were isolated from phagocytic (monocytes/macrophages)and non-phagocytic (T-lymphocytes) cells obtained from mice bearingLNCaP and LS174T tumors were selected for these studies since the formercell line expresses PSA (Denmeade et al. (2001) Prostate 48:1; Lin etal. (2001) J. Urol. 166:1943) and the latter exhibits a tumor-specificglycoprotein (TAG-72), a high molecular weight mucin (Colcher et al.(1981) Proc. Natl. Acad. Sci. USA 78:3199); Colcher et al. (1984) CancerRes. 44:5744; Kassis et al. (1996) J. Nucl. Med. 37:343. Western blotanalysis was carried out with 16 μg of the purified protein samples. Inessence, each sample was mixed with two volumes of SDS loading bufferand run on 10% SDS-PAGE along with unstained precision plus proteinstandards (Biorad) in Tris-glycine-SDS buffer (pH 8.4) at 200 volts. Theproteins were transferred to a nitrocellulose membrane (overnight at 4°C.) using a Mini Trans-Blot (Biorad) apparatus and a transfer buffercontaining 25 mM Tris, pH 8.4, 192 mM glycine, and 20% methanol. Themembrane was blocked with 5% nonfat dry milk (60 min at room temperature(RT)) and incubated (1 hour, RT) with either B72.3, a mouse monoclonalantibody against human TAG-72, or ER-PR8, a mouse monoclonal antibodyagainst human PSA. The blots were washed and then incubated withImmun-Star Goat Anti Mouse-HRP conjugate (Biorad), a secondary antibodyspecific to mouse IgG, and developed by incubation (5 min, RT) with a1:1 mixture of luminol solution and peroxide buffer (Biorad), followedby autoradiography.

The data clearly indicated that phagocytic cells from LNCaPtumor-bearing mice were positive for PSA, whereas this protein could notbe detected in non-phagocytic T cells from the same animals as shown inFIG. 24. Similarly, TAG-72 was expressed by monocytes/macrophagesobtained from LS174T tumor-bearing mice and was completely absent in Tcells from the same animals. These findings demonstrate the“acquisition” and expression of tumor-specific protein signatures byphagocytic cells.

While these data are specific to animals with cancer and phagocytic andnon-phagocytic cells obtained from the blood of mice, the describedmethods are also useful in humans and in the diagnosis and/or detectionof one or more other disorders and/or diseases and with phagocytic andnon-phagocytic cells obtained from other bodily fluids.

Example 10 Profiling Experiments

Isolation of Blood Phagocytic Cells

A sample of blood is obtained from a patient. The blood (˜5 mL) will betransferred to a 50-mL tube containing 50 μL 0.5 M EDTA (final EDTAconcentration=˜4.8 mM). The tube will be vortexed gently and 25 mL RBCLysis Buffer (Norgen, Incorporated) will be added. The tube will bevortexed gently again, incubated at room temperature until the color ofthe solution changes to bright red (3-5 min), and centrifuged at 2,000rpm for 3 min. Following careful aspiration of the supernatant, the WBCswill be washed with 40 mL Ca/Mg-free 0.1 M PBS (containing 2% FBS, 2 mMEDTA, and 20 mM glucose), and the cells (10⁶/mL) will then be incubated(30 min, 4° C., in the dark) with a cell-staining solution containing(i) the DNA, viable cell-permeable stain Hoechst 33342 (4 g/mL; Em=483nm), (ii) the anti-human monocytes/macrophages monoclonal antibody(Alexa Fluor® 647-conjugate; Em=668 nm), which recognizes the humanF4/80 antigen expressed by circulating monocytes/macrophages, and (iii)the anti-human neutrophil monoclonal antibody (RPE-conjugate; Em=578nm), which recognizes human circulating neutrophils. The cells will thenbe washed and sorted (BD FACSAria) into neutrophils (N_(n=2)),neutrophils (N_(n>2)), monocytes/macrophages (M/M_(n=2)), andmonocytes/macrophages (M/M_(n>2)).

Gene Profiling

Human whole-genome gene profiling will be performed. For RNA samplesobtained from human tumor cells or neutrophils (N_(n=2), N_(n>2)) andmonocytes/macrophages (M/M_(n=2), M/M_(n>2)), the GeneChip® Human GenomeU133 Plus 2.0 Array by Affymetrix, Incorporated will be used. This arrayanalyzes the expression level of over 47,000 transcripts and variants,including 38,500 well-characterized human genes. In general, theextracted RNA will be used to determine the expression profiles of humangenes using the above-mentioned array. To ensure array reproducibility,each sample will be profiled in triplicate and the experiment repeatedonce. The microarray data will be filtered for cancer-induction-relatedgenes as described below and validated using quantitative real-time,reverse transcriptase, polymerase chain reaction (RT-PCR).

Upregulation/Downregulation of Cancer-Induction-Related Genes

RNA will be isolated using Triazol (Invitrogen, Incorporated) andpurified using the cartridges provided in the kit. The RNA quality andquantity will be assessed with the Bioanalyzer 2100 (AgilentTechnologies, Incorporated, Palo Alto, Calif.) and Degradometer softwareversion 1.41 (Worldwide Web: dnaarrays.org). These experimental resultswill help in distinguishing the molecular pathways perturbed consequentto the presence of tumors.

Analysis of Microarray Experiments

The analysis of the large scale/high throughput molecular expressiondata generated will rely heavily on the ability to (i) identify genesdifferentially expressed in phagocytic cells with a DNA content >2, (ii)annotate the identified genes, and (iii) assign the annotated genes tothose specifically expressed by a specific tumors. Statistical analysisof the microarray data can be done, for example, using the dChip packagewhich easily accommodates this type of gene list construction in its“Analysis/Compare Samples” menu. When using Affymetrix GeneChips, one ormore Gene Chips and associated methods will be applied to ascertain thequality of the raw microarray data (Gautier et al. (2004) Bioinformatics20:307). Furthermore, various background correction and normalizationprocedures will be utilized to arrive at an optimal protocol fornormalization and summarization of the probe sets (to produce expressionvalues) (Huber et al. (2002) Bioinformatics 18 (Suppl. 1):596; Wu et al.(2004) Journal of the American Statistical Association 99:909; Seo andHoffman (2006) BioMed Central Bioinformatics 7:395). In a two-stepfiltration approach, we will compare the gene profiles of P_(n=2) tothose of P_(n>2) and construct a list of expressed genes and thencompare these genes to the tumor-specific genes identified for eachtumor cell line—post filtration of P_(n=2) gene profile as shown in FIG.5. For example, (i) blood will be obtained from breast cancer patients;(ii) neutrophils (n>2 and n=2) will be isolated and their gene profilesdetermined in triplicate; (iii) the mean (from the 3 samples) of eachidentified gene and its respective standard error (SE) will becalculated for each group (N_(n>2) and N_(n=2)); (iv) the geneexpression profiles of the two groups will then be compared and a list(L-1) of expressed genes identified on the basis of an absolute ≧2-foldlog change (N_(n>2)/N_(n=2)), according to the Welch modified two-samplet-test; (v) the gene expression profiles of N_(n=2) and that of breastcancer (obtained from tumor and normal breast tissue biopsies) will becompared and a list (L-2) of expressed genes identified; and (vi)breast-cancer-specific gene signatures that have been acquired/expressedby N_(n>2) will be identified by comparing the genes in L-1 and L-2(“Analysis/Compare Samples/Combine Comparisons,” dChip) and filteringcommon genes.

Protein Profiling

Fifty to one hundred micrograms of the total protein from each type ofcells will be denatured and reduced withtris-(2-carboxyethyl)phosphinetrypsin (1 mM) and 0.02% sodium dodecylsulfate at 60° C. for 1 hour. Cysteines are subsequently blocked andtotal protein is digested with trypsin at 37° C. for 12-16 hours. Theresulting peptides will be iTRAQ-labeled (with tags 113-119 and 121) for1 hour (4-plex or 8-plex depending on the number of cell types to becompared). Following labeling, the separately tagged samples arecombined and injected into an Agilent 1200 Series HPLC system equippedwith a strong cation exchange column (Applied Biosystems 4.6×100Porous). The 96 collected fractions are then pooled into 14 fractions,and each fraction is injected into the LC Packings Ultimate HPLC Systemfor a second round of fractionation under reverse-phase conditions (LCPackings 15 cm×75 am analytical column). The reverse-phase fractions arespotted directly onto the target plate using an LC Packings Probot andare analyzed with mass spectrometry (Applied Biosystems 4800 PlusProteomics Analyzer). Following data acquisition, the spectra areprocessed using the ProteinPilot software package (Applied BiosystemsMDS Sciex), and the individual proteins in each of the cell types withtheir relative expression levels are identified using the ProteinPilot™software (the analysis and identification of cancer-associated proteomicsignatures will be similar to that outlined in FIG. 5 for the genomicsignatures).

What is claimed:
 1. A method for diagnosing the presence of a cancercell in an individual comprising the steps of: obtaining a firstexpression profile from a blood phagocytic cell from an individual;obtaining a second expression profile from a blood non-phagocytic cellfrom the individual; comparing the first and second expression profiles;identifying differential expression of one or more markers specific tothe first expression profile; and relating the differential expressionof the one or more markers specific to the first expression profile tothe presence of a cancer cell in the individual.
 2. The method of claim1, wherein the one or more markers are selected from the groupconsisting of DNA, RNA, protein, lipid, carbohydrate and combinationsthereof.
 3. The method of claim 1, wherein the blood phagocytic cell isselected from the group consisting of one or more of a neutrophil, amacrophage, a monocyte, a dendritic cell and a foam cell.
 4. The methodof claim 1, wherein the blood non-phagocytic cell is selected from thegroup consisting of one or more of a T cell, a B cell, a null cell and abasophil.
 5. The method of claim 1, wherein the blood phagocytic celland the blood non-phagocytic cell are isolated from whole blood, urine,stool, saliva, lymph or cerebrospinal fluid.
 6. The method of claim 5,wherein the blood phagocytic cell and the blood non-phagocytic cell areisolated using antibodies.
 7. The method of claim 5, wherein the bloodphagocytic cell and the blood non-phagocytic cell are separated byfluorescence activated cell sorting.
 8. The method of claim 5, whereinthe blood phagocytic cell and the blood non-phagocytic cell areseparated using a ligand that binds to a molecular receptor expressed onthe plasma membranes of WBC populations.
 9. The method of claim 5,wherein the blood phagocytic cell and the blood non-phagocytic cell areseparated by one or methods selected from the group consisting offiltration, gradient-based centrifugation, elution, microfluidics. 10.The method of claim 1, wherein the blood phagocytic cell and the bloodnon-phagocytic cell are isolated from a population of white blood cells.11. The method of claim 10, wherein the blood phagocytic cell and theblood non-phagocytic cell are isolated using antibodies.
 12. The methodof claim 10, wherein the blood phagocytic cell and the bloodnon-phagocytic cell are separated by one or more methods selected fromthe group consisting of fluorescence activated cell sorting, filtration,gradient-based centrifugation, elution and microfluidics.
 13. The methodof claim 10, wherein the blood phagocytic cell and the bloodnon-phagocytic cell are separated using a ligand that binds to molecularreceptors expressed on the plasma membranes of WBC populations.
 14. Themethod of claim 1, wherein the individual has one or more of occultcancer, previously diagnosed primary cancer and metastatic cancer. 15.The method of claim 1, further comprising the step of relating thepresence of one or more markers to efficacy of a cancer therapy.
 16. Themethod of claim 1, wherein the marker is selected from one or more ofDNA, RNA and microRNA corresponding one or more of a cancer gene, anoncogene and a tumor suppressor gene.
 17. The method of claim 1, whereinthe marker is one or both of a protein and a polypeptide encoded by oneor more of a cancer gene, and oncogene and a tumor suppressor gene. 18.A method for identifying a tumor-specific signature in an individualhaving cancer comprising the steps of: obtaining a first expressionprofile from a blood phagocytic cell from an individual having cancer;obtaining a second expression profile from a blood non-phagocytic cellfrom the individual having cancer; comparing the first and secondexpression profiles; identifying differential expression of two or moremarkers specific to the first expression profile; and relating thedifferential expression of the two or more markers specific to atumor-specific signature in the individual having cancer.
 19. The methodof claim 18, wherein the two or more markers are selected from the groupconsisting of DNA, RNA, protein, lipid, carbohydrate and combinationsthereof.
 20. The method of claim 18, wherein the two or more markers areDNA or RNA corresponding to two or more cancer genes, oncogenes, tumorsuppressor genes or combinations thereof.
 21. The method of claim 18,wherein the two or more markers are proteins or polypeptides encoded bytwo or more cancer genes, oncogenes, tumor suppressor genes orcombinations thereof.
 22. A method for diagnosing the presence of acancer cell in an individual comprising the steps of: obtaining a firstexpression profile from a blood phagocytic cell from an individual;obtaining a second expression profile from a blood non-phagocytic cellfrom the individual; comparing the first and second expression profiles;identifying the presence of a circulating tumor cell or subcellularfragment thereof specific to the first expression profile; and relatingthe presence of a circulating tumor cell or subcellular fragment thereofto the presence of a cancer cell in the individual.
 23. The method ofclaim 22, wherein an increase in the quantity of a marker in the firstexpression profile relative to the second expression profile indicatesthe presence of one or both of a circulating tumor cell and asubcellular fragment thereof.
 24. The method of claim 23, wherein themarker is selected from the group consisting of DNA, RNA, protein,lipid, carbohydrate and combinations thereof.
 25. The method of claim23, wherein the marker is selected from the group consisting of DNA,RNA, microRNA and combinations thereof corresponding to a cancer gene,an oncogene, a tumor suppressor gene or a combination thereof.
 26. Themethod of claim 23, wherein the marker is a protein or polypeptideencoded by a cancer gene, an oncogenes, a tumor suppressor gene or acombination thereof.
 27. A method for diagnosing the presence of acancer cell in an individual comprising the steps of: isolating apopulation of phagocytic cells from an individual; separating 2nphagocytic cells from >2n phagocytic cells; obtaining a first expressionprofile from the 2n phagocytic cells; obtaining a second expressionprofile from the >2n phagocytic cells; comparing the first and secondexpression profiles; identifying differential expression of one or moremarkers specific to the first expression profile; and relating thedifferential expression of the one or more markers specific to the firstexpression profile to the presence of a cancer cell in the individual.28. The method of claim 27, wherein the one or more markers are selectedfrom the group consisting of DNA, RNA, protein, lipid, carbohydrate andcombinations thereof.
 29. The method of claim 27, wherein the one ormore markers are selected from the group consisting of DNA, RNA,microRNA and combinations thereof corresponding to two or more cancergenes, oncogenes, tumor suppressor genes or combinations thereof. 30.The method of claim 27, wherein the one or more markers are proteins orpolypeptides encoded by two or more cancer genes, oncogenes, tumorsuppressor genes or any combination thereof.
 31. The method of claim 27,wherein the blood phagocytic cell is selected from the group consistingof a neutrophil, a macrophage, a monocyte, a dendritic cell, a foam celland any combination thereof.
 32. The method of claim 27, wherein theblood phagocytic cell is isolated from whole blood, urine, stool,saliva, lymph or cerebrospinal fluid.
 33. The method of claim 32,wherein the blood phagocytic cell is isolated using antibodies.
 34. Themethod of claim 32, wherein the blood phagocytic cell is separated usingone or methods selected from the group consisting of fluorescenceactivated cell sorting, filtration, gradient-based centrifugation,elution and microfluidics.
 35. The method of claim 32, wherein the bloodphagocytic cell is separated using a ligand that binds to a molecularreceptor expressed on the plasma membranes of WBC populations.
 36. Amethod for diagnosing the presence of an infectious agent in anindividual comprising the steps of: obtaining a first expression profilefrom a blood phagocytic cell from an individual; obtaining a secondexpression profile from a blood non-phagocytic cell from the individual;comparing the first and second expression profiles; identifyingdifferential expression of one or more markers specific to the firstexpression profile; and relating the differential expression of the oneor more markers specific to the first expression profile to the presenceof an infectious agent in the individual.
 37. The method of claim 36,wherein the one or more markers are selected form the group consistingof pathogen DNA, pathogen RNA, pathogen protein, pathogen polypeptide,pathogen lipid and combinations thereof.
 38. The method of claim 36,wherein the infectious agent is selected from the group consisting of avirus, a bacterium, a fungus, a parasite and an infectious protein. 39.A method for identifying an infectious agent-specific signature in aninfected individual comprising the steps of: obtaining a firstexpression profile from a blood phagocytic cell from an infectedindividual; obtaining a second expression profile from a bloodnon-phagocytic cell from the infected individual; comparing the firstand second expression profiles; identifying differential expression oftwo or more markers specific to the first expression profile; andrelating the differential expression of the two or more markers specificto an infectious agent-specific signature in the infected individual.40. The method of claim 39, wherein the two or more markers are selectedform the group consisting of pathogen DNA, pathogen RNA, pathogenprotein, pathogen polypeptide, pathogen lipid and combinations thereof.41. The method of claim 39, wherein the infectious agent is selectedfrom the group consisting of a virus, a bacterium, a fungus, a parasiteand an infectious protein.
 42. A method for diagnosing the presence ofan infectious agent in an individual comprising the steps of: obtaininga first expression profile from a blood phagocytic cell from anindividual; obtaining a second expression profile from a bloodnon-phagocytic cell from the individual; comparing the first and secondexpression profiles; identifying the presence of a circulating tumorcell or subcellular fragment thereof specific to the first expressionprofile; and relating the presence of a circulating tumor cell orsubcellular fragment thereof to the presence of an infectious agent inthe individual.
 43. A method for diagnosing the presence of aninfectious agent in an individual comprising the steps of: isolating apopulation of phagocytic cells from an individual; separating 2nphagocytic cells from >2n phagocytic cells; obtaining a first expressionprofile from the 2n phagocytic cells; obtaining a second expressionprofile from the >2n phagocytic cells; comparing the first and secondexpression profiles; identifying differential expression of one or moremarkers specific to the first expression profile; and relating thedifferential expression of the one or more markers specific to the firstexpression profile to the presence of an infectious agent in theindividual.
 44. The method of claim 43, wherein the one or more markersare selected form the group consisting of pathogen DNA, pathogen RNA,pathogen protein, pathogen polypeptide, pathogen lipid and combinationsthereof.
 45. The method of claim 43, wherein the infectious agent isselected from the group consisting of a virus, a bacterium, a fungus, aparasite and an infectious protein.
 46. The method of claim 43, whereinthe blood phagocytic cell is selected from the group consisting of aneutrophil, a macrophage, a monocyte, a dendritic cell, a foam cell andany combination thereof.
 47. The method of claim 43, wherein the bloodphagocytic cell is isolated from whole blood, urine, stool, saliva,lymph or cerebrospinal fluid.
 48. The method of claim 47, wherein theblood phagocytic cell is isolated using antibodies.
 49. The method ofclaim 47, wherein the blood phagocytic cell is separated using one ormethods selected from the group consisting of fluorescence activatedcell sorting, filtration, gradient-based centrifugation, elution andmicrofluidics.
 50. The method of claim 47, wherein the blood phagocyticcell is separated using a ligand that binds to a molecular receptorexpressed on the plasma membranes of WBC populations.
 51. One or moremarkers of a condition differentially expressed between phagocytic cellsand nonphagocytic cells from a common source.
 52. One or more markers ofa condition differentially expressed between phagocytic cells having aDNA content of 2n and phagocytic cells having a DNA content of greaterthan 2n from a common source.
 53. The one or more markers of claim 51being DNA, RNA, microRNA, protein, lipid, or carbohydrate orcombinations thereof.
 54. The one or more markers of claim 52 being DNA,RNA, microRNA, protein, lipid, or carbohydrate or combinations thereof.55. The one or more markers of claim 51 wherein the phagocytic cells areneutrophils, macrophages, monocytes, dendritic cells or foam cells orcombinations thereof.
 56. The one or more markers of claim 52 whereinthe phagocytic cells are neutrophils, macrophages, monocytes, dendriticcells or foam cells or combinations thereof.
 57. The one or more markersof claim 51 wherein the nonphagocytic cells are T cells, B cells, nullcells, or basophils or combinations thereof.
 58. Protein profile, DNAprofile, RNA profile, metabolite profile, glycome profile, glycoproteinprofile, carbohydrate profile, lipoprotein profile or lipid profilespecific to blood phagocytic cells compared to blood nonphagocytic cellsfrom a common source.
 59. Protein profile, DNA profile, RNA profile,metabolite profile, glycome profile, glycoprotein profile, carbohydrateprofile, lipoprotein profile or lipid profile specific to bloodphagocytic cells having a DNA content of greater than 2n compared toblood phagocytic cells having a DNA content of 2n from a common source.60. A kit for detecting the presence of one or more markers associatedwith cancer and/or an infectious agent in a biological sample, whereinthe kit comprises a labeled compound or agent for detecting a markerpolypeptide, protein lipid, oligosaccharide, mRNA, microRNA, or genomicDNA, and a standard such as a non-phagocytic cell or a 2n cell tocompare the amount of marker in the sample with the standard.